Caninized anti-ngf antibodies and methods thereof

ABSTRACT

The invention provides novel caninized anti-NGF antibodies (such as caninized anti-NGF antagonist antibodies and antigen binding proteins), and polynucleotides encoding the same. The invention further provides use of said antibodies or antigen binding proteins and/or nucleotides in the treatment and/or prevention of NGF related disorders, particularly pain.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application No.61/656,056 filed Jun. 6, 2012 which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunology. Morespecifically, the present invention relates to anti-NGF antigen bindingproteins or antibodies that have been modified to become non-immunogenicin canines, particularly chimeric antibodies and caninized antibodiesthat specifically bind to canine NGF. The invention further concerns useof such antigen binding proteins or antibodies in the treatment and/orprevention of NGF related disorders, particularly pain, in canines.

BACKGROUND OF THE INVENTION

Nerve growth factor (NGF) was the first neurotrophin to be identified,and its role in the development and survival of both peripheral andcentral neurons has been well characterized. NGF has been shown to be acritical survival and maintenance factor in the development ofperipheral sympathetic and embryonic sensory neurons and of basalforebrain cholinergic neurons (Smeyne, et al., Nature 368:246-249 (1994)and Crowley, et al., Cell 76: 1001-101 I (1994)). NGF upregulatesexpression of neuropeptides in sensory neurons (Lindsay, et al, Nature337:362-364 (1989)), and its activity is mediated through two differentmembrane-bound receptors, the TrkA tyrosine kinase receptor and the p75common neurotrophin receptor (sometimes termed “high affinity” and “lowaffinity” NGF receptors, respectively) which is structurally related toother members of the tumor necrosis factor receptor family (Chao, etal., Science 232:518-521 (1986)).

In addition to its effects in the nervous system, NGF has beenincreasingly implicated in processes outside of the nervous system. Forexample, NGF has been shown to enhance vascular permeability (Otten, etal., Eur J Pharmacol. 106: 199-201 (1984)), enhance T- and B-cell immuneresponses (Otten, et al., Proc. Natl. Acad. Sci. USA 86:10059-10063(1989)), induce lymphocyte differentiation and mast cell proliferationand cause the release of soluble biological signals from mast cells(Matsuda, et al., Proc. Natl. Acad. Sci. USA 85:6508-6512 (1988);Pearce, et al., J. Physiol. 372:379-393 (1986); Bischoff, et al., Blood79:2662-2669 (1992); Horigome, et al., J. Bioi. Chem. 268:14881-14887(1993)).

NGF is produced by a number of cell types including mast cells (Leon, etal., Proc. Natl. Acad. Sci. USA 91:3739-3743 (1994)), B-lymphocytes(Torcia, et al., Cell 85:345-356 (1996), keratinocytes (Di Marco, etal., J. Biol. Chem. 268: 22838-22846)), smooth muscle cells (Ueyama, etal., J. Hypertens. 11: 1061-1065 (1993)), fibroblasts (Lindholm, et al.,Eur. J. Neurosci. 2:795-801 (1990)), bronchial epithelial cells (Kassel,et al., Clin, Exp. Allergy 31:1432-40 (2001)), renal mesangial cells(Steiner, et al., Am. J. Physiol. 261: F792-798 (1991)) and skeletalmuscle myotubes (Schwartz, et al., J Photochem. Photobiol. B66: 195-200(2002)). NGF receptors have been found on a variety of cell typesoutside of the nervous system. For example, TrkA has been found on humanmonocytes, T- and B-lymphocytes and mast cells.

An association between increased NGF levels and a variety ofinflammatory conditions has been observed in human patients as well asin several animal models. These include systemic lupus erythematosus(Bracci-Laudiero, et al., Neuroreport 4:563-565 (1993)), multiplesclerosis (BracciLaudiero, et al, Neurosci. Lett. 147:9-12 (1992)),psoriasis (Raychaudhuri, et al., Acta Dern. l′enereol. 78:84-86 (1998)),arthritis (Falcim, et al., Ann. Rheum. Dis. 55:745-748 (1996)),interstitial cystitis (Okragly, et al., J. Urology 161: 438-441 (1999))and asthma (Braun, et al., Eur. J Immunol. 28:3240-3251 (1998)).

Consistently, an elevated level of NGF in peripheral tissues isassociated with hyperalgesia and inflammation and has been observed in anumber of forms of arthritis. The synovium of patients affected byrheumatoid arthritis expresses high levels of NGF while in non-inflamedsynovium NGF has been reported to be undetectable (Aloe, et al., Arch.Rheum. 35:351-355 (1992)). Similar results were seen in rats withexperimentally induced rheumatoid arthritis (Aloe, et al., Clin. Exp.Rheumatol. 10:203-204 (1992)). Elevated levels of NGF have been reportedin transgenic arthritic mice along with an increase in the number ofmast cells (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects15:139-143 (1993)).

Osteoarthritis (OA) is one of the most common chronic musculoskeletaldiseases in dogs, affecting 20% of the canine population over one yearof age. The development of OA is mainly secondary to trauma, jointinstability, and diseases such as hip dysplasia. Osteoarthritis is adisease condition of the entire joint, and both inflammatory anddegenerative changes of all articular structures result in disabilityand clinical signs of lameness and pain. Pain is the most importantclinical manifestation of canine OA and it is the result of a complexinterplay between structural joint changes, biochemical and molecularalterations, as well as peripheral and central pain-processingmechanisms. Within this network, the activation and sensitization ofperipheral nociceptors by inflammatory and hyperalgesic mediators (e.g.cytokines, prostaglandins and neuromediators) is one of the mainperipheral mechanisms responsible for the joint pain. Nerve growthfactor (NGF) is one of the neuromediators that has received broaderattention as a key regulator involved in both inflammatory andneuropathic pain. (Isola et al. Vet Comp Orthop Traumatol 4: 2011 pgs279-284)

SUMMARY OF THE INVENTION

The invention provides a novel caninized anti-NGF antigen bindingprotein (such as caninized anti-NGF antagonist antibodies), andpolynucleotides encoding the same. The invention further provides use ofsaid antigen binding proteins and/or nucleotides in the treatment and/orprevention of NGF related disorders, particularly pain.

In one embodiment the present invention provides an isolated antigenbinding protein or antibody fragment that specifically binds to canineNGF. In some embodiments the antigen binding protein is selected fromthe group consisting of: a monoclonal antibody, a chimeric antibody, asingle chain antibody, a tetrameric antibody, a tetravalent antibody, amultispecific antibody, a domain-specific antibody, a domain-deletedantibody, a fusion protein, an ScFc fusion protein, an Fab fragment, anFab′ fragment, an F(ab′)₂ fragment, an Fv fragment, an ScFv fragment, anFd fragment, a single domain antibody, a dAb fragment, a small modularimmunopharmaceutical (SMIP) a nanobody, and IgNAR molecule. In someembodiments said antibody is a monoclonal antibody. In some embodimentssaid antibody is chimeric. In one or more embodiments the antigenbinding protein of the invention is caninized. In one or moreembodiments the antigen binding protein of the invention is a canineantibody.

In one or more embodiments the antigen binding protein that specificallybinds to canine NGF prevents canine NGF from binding to canine TrkA andto a lesser extent p75 thus inhibiting signaling through canine TrkA andp75, which has been shown to reduce the signaling through sensoryneurons thus reducing levels of pain. In one or more embodiments, theantigen binding protein of the invention has no significant adverseeffect on the immune system of a canine.

In one or more embodiments the isolated antigen binding protein thatspecifically binds to canine NGF treats an NGF related disorder in acanine. In some embodiments the NGF related disorder in a canine and isselected from the group consisting of: cardiovascular diseases,atherosclerosis, obesity, diabetes, metabolic syndrome, pain andinflammation. In some embodiments the NGF related disorder is pain. Insome embodiments the type of pain is selected from the group consistingof: chronic pain; inflammatory pain, post-operative incision pain,neuropathic pain, fracture pain, osteoporotic fracture pain,post-herpetic neuralgia, cancer pain, pain resulting from burns, painassociated with wounds, pain associated with trauma, neuropathic pain,pain associated with musculoskeletal disorders such as rheumatoidarthritis, osteoarthritis, ankylosing spondylitis, seronegative(non-rheumatoid) arthropathies, non-articular rheumatism andperiarticular disorders and peripheral neuropathy. In some embodimentsthe type of pain is chronic pain. In some embodiments, the type of painis osteoarthritis pain.

In one or more embodiments, the present invention provides an isolatedantigen binding protein and comprising at least one selected from thegroup consisting of:

a variable heavy chain (VH) comprising a complementary determiningregion (CDR 1) having the amino acid sequence LIGYDIN (SEQ ID NO.1);LIQYDIN (SEQ ID NO. 7) or LIEYDIN (SEQ ID NO. 8);

a variable heavy chain (VH) comprising a complementary determiningregion (CDR2) having the amino acid sequence MIWGDGTTDYNSALKS (SEQ IDNO.2); or MIWGTGTTDYNSALKS (SEQ ID NO.13);

a variable heavy chain (VH) comprising a complementary determiningregion (CDR3) having the amino acid sequence GGYYYGTSYYFDY (SEQ IDNO.3); or GGYWYATSYYFDY (SEQ ID NO. 9); and

a variant thereof having one or more conservative amino acidsubstitutions in at least one of CDR1, CDR2 or CDR3.

In one or more embodiments, the present invention provides an isolatedantigen binding protein comprising at least one selected from the groupconsisting of:

a variable heavy chain (VL) comprising a complementary determiningregion (CDR 1) having the amino acids sequence RASQDISNHLN (SEQ ID NO.4); or RASQSISNNLN (SEQ ID NO. 10);

a variable heavy chain (VL) comprising a complementary determiningregion (CDR 2) having the amino acids sequence YISRFHS (SEQ ID NO. 5) orYISSFHS (SEQ ID NO. 11);

a variable heavy chain (VL) comprising a complementary determiningregion (CDR 3) having the amino acids sequence QQSKTLPYT (SEQ ID NO.6)or QQSHTLPYT (SEQ ID NO. 12); and

a variant thereof having one or more conservative amino acidsubstitutions in at least one of CDR1, CDR2 or CDR3. In some embodimentsthe present invention provides a isolated antigen binding protein havingat least one of the variable light chain CDRs described above, and canfurther include at least one of the following variable heavy chain CDRsselected from the group consisting of:

a variable heavy chain (VH) comprising a complementary determiningregion (CDR 1) having the amino acid sequence LIGYDIN (SEQ ID NO.1);LIQYDIN (SEQ ID NO. 7) or LIEYDIN (SEQ ID NO. 8);

a variable heavy chain (VH) comprising a complementary determiningregion (CDR2) having the amino acid sequence MIWGDGTTDYNSALKS (SEQ IDNO.2); or MIWGTGTTDYNSALKS (SEQ ID NO.13);

a variable heavy chain (VH) comprising a complementary determiningregion (CDR3) having the amino acid sequence GGYYYGTSYYFDY (SEQ IDNO.3); or GGYWYATSYYFDY (SEQ ID NO. 9); and

a variant thereof having one or more conservative amino acidsubstitutions in at least one of CDR1, CDR2 or CDR3.

In one or more embodiments, the antigen binding protein of the presentinvention may include at least one of the following:

a variable heavy chain comprising:

QVQLKESGPGLVAPSQSLSITCTVSGFSLIGYDINWVRQPPGKGLEWLGMIWGDGTTDYNSALKSRLSISKDNSKSQVFLKMNSLRTDDTATYSCARGGYYYGTSYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD (SEQ ID NO. 14; MU-RN911-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIGYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYYYGTSYYFDYWGQGTLVTVSS (SEQ ID NO: 17; CAN-N2G9-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIGYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 25; CAN-LTM109-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIQYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 27; CAN-SSM57-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIEYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 28; CAN-SSM58-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIEYDINWVRQAPGKGLQWVTMIWGTGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 29; CAN-SSM66-VH);

and variants thereof having one or more conservative amino acidsubstitutions.

In one or more embodiments, the isolated antigen binding protein of theinvention may include at least one of the following:

a variable light chain comprising:

DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGGGTKLEIKRA (SEQ ID NO. 15; MU-RN911-VL);DIVMTQTPLSLSVSPGETASISCRASQDISNHLNWFRQKPGQSPQRLIYYISRFHSGVPDRFSGGSGTDFTLRISRVEADDTGVYYCQQSKTLPYTFGAGTKLEIK (SEQ ID NO. 16; CAN-E3M-VL));DIVMTQTPLSLSVSPGEPASISCRASQDISNHLNWFRQKPGQSPQRLIYYISRFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSKTLPYTFGQGTKLEIK (SEQ ID NO. 18; CAN-618-VL));DIVMTQTPLSLSVSPGEPASISCRASQDISNHLNWFRQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSKTLPYTFGQGTKLEIK (SEQ ID NO. 19; CAN-QC23-VL));DIVMTQTPLSLSVSPGEPASISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGGGTKLEI (SEQ ID NO. 20; CAN-618FW1-VL);DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWFRQKPGQSPQRLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGGGTKLEI (SEQ ID NO. 21; CAN-618FW2-VL);DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSKTLPYTFGGGTKLEI (SEQ ID NO. 22; CAN-618FW3-VL);DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGQGTKLEI (SEQ ID NO. 23; CAN-618FW4-VL);DIVMTQTPLSLSVSPGETASISCRASQSISNNLNWFRQKPGQSPQRLIYYISRFHSGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQSHTLPYTFGAGTKLEIK (SEQ ID NO. 24; CAN-LTM109-VL);DIVMTQTPLSLSVSPGETASISCRASQSISNNLNWFRQKPGQSPQRLIYYISSFHSGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQSHTLPYTFGAGTKLEIK (SEQ ID NO. 26; CAN-SSME3M-VL);DIVMTQTPLSLSVSPGEPASISCRASQSISNNLNWFRQKPDGTVKLLIYYISSFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSHTLPYTFGQGTKLEIK (SEQ ID NO. 30; CAN-SSMQC23-VL); ,and

variants thereof having one or more conservative amino acidsubstitutions. In some embodiments the present invention provides anisolated antigen binding protein having at least one of the variablelight chain described above, and can further include at least one of thefollowing variable heavy chain of the isolated antibody or antigenbinding portion thereof selected from the group consisting of:

QVQLKESGPGLVAPSQSLSITCTVSGFSLIGYDINWVRQPPGKGLEWLGMIWGDGTTDYNSALKSRLSISKDNSKSQVFLKMNSLRTDDTATYSCARGGYYYGTSYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD (SEQ ID NO. 14; MU-RN911-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIGYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYYYGTSYYFDYWGQGTLVTVSS (SEQ ID NO: 17; CAN-N2G9-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIGYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 25; CAN-LTM109-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIQYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 27; CAN-SSM57-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIEYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 28; CAN-SSM58-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIEYDINWVRQAPGKGLQWVTMIWGTGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 29; CAN-SSM66-VH);

and variants thereof having one or more conservative amino acidsubstitutions.

In one or more embodiments, the isolated antigen binding protein of theinvention can include at least one of the following:

a) a variable light chain comprising:

DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGGGTKLEIKRA (SEQ ID NO. 15; MU-RN911-VL);DIVMTQTPLSLSVSPGETASISCRASQDISNHLNWFRQKPGQSPQRLIYYISRFHSGVPDRFSGGSGTDFTLRISRVEADDTGVYYCQQSKTLPYTFGAGTKLEIK (SEQ ID NO. 16; CAN-E3M-VL));DIVMTQTPLSLSVSPGEPASISCRASQDISNHLNWFRQKPGQSPQRLIYYISRFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSKTLPYTFGQGTKLEIK (SEQ ID NO. 18; CAN-618-VL));DIVMTQTPLSLSVSPGEPASISCRASQDISNHLNWFRQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSKTLPYTFGQGTKLEIK (SEQ ID NO. 19; CAN-QC23-VL));DIVMTQTPLSLSVSPGEPASISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGGGTKLEI (SEQ ID NO. 20; CAN-618FW1-VL);DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWFRQKPGQSPQRLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGGGTKLEI (SEQ ID NO. 21; CAN-618FW2-VL);DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSKTLPYTFGGGTKLEI (SEQ ID NO. 22; CAN-618FW3-VL);DIQMTQTTSSLSASLGDRVTISCRASQDISNHLNWYQQKPDGTVKLLIYYISRFHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQSKTLPYTFGQGTKLEI (SEQ ID NO. 23; CAN-618FW4-VL);DIVMTQTPLSLSVSPGETASISCRASQSISNNLNWFRQKPGQSPQRLIYYISRFHSGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQSHTLPYTFGAGTKLEIK (SEQ ID NO. 24; CAN-LTM109-VL);DIVMTQTPLSLSVSPGETASISCRASQSISNNLNWFRQKPGQSPQRLIYYISSFHSGVPDRFSGSGSGTDFTLRISRVEADDTGVYYCQQSHTLPYTFGAGTKLEIK (SEQ ID NO. 26; CAN-SSME3M-VL);DIVMTQTPLSLSVSPGEPASISCRASQSISNNLNWFRQKPDGTVKLLIYYISSFHSGVPSRFSGSGSGTDFTLRISRVEADDAGVYYCQQSHTLPYTFGQGTKLEIK (SEQ ID NO. 30; CAN-SSMQC23-VL); ,and variants thereof having one or more conservative amino acidsubstitutions; and b) a variable heavy chain comprising:

QVQLKESGPGLVAPSQSLSITCTVSGFSLIGYDINWVRQPPGKGLEWLGMIWGDGTTDYNSALKSRLSISKDNSKSQVFLKMNSLRTDDTATYSCARGGYYYGTSYYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD (SEQ ID NO. 14; MU-RN911-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIGYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYYYGTSYYFDYWGQGTLVTVSS (SEQ ID NO: 17; CAN-N2G9-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIGYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 25; CAN-LTM109-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIQYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 27; CAN-SSM57-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIEYDINWVRQAPGKGLQWVTMIWGDGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 28; CAN-SSM58-VH);EVQLVESGGDLARPGGSLKLSCVVSGFSLIEYDINWVRQAPGKGLQWVTMIWGTGTTDYNSALKSRFTVSRDNAMNTVYLQMNSLRVEDTAVYYCARGGYWYATSYYFDYWGQGTLVTVSS (SEQ ID NO: 29; CAN-SSM66-VH);

and variants thereof having one or more conservative amino acidsubstitutions.

In one or more embodiments, the present invention provides an isolatedantigen binding protein wherein the variable light chain comprises SEQID NO. 16 (CAN-E3M-VL) and the variable heavy chain comprises SEQ ID NO.17 (CAN-N2G9-VH).

In one or more embodiments, the present invention provides an isolatedantigen binding protein comprising a variable light chain comprising SEQID NO. 26 (CAN-SSME3M-VL) and the variable heavy chain comprising SEQ IDNO. 27 (CAN-SSM57-VH).

In one or more embodiments, the present invention provides an isolatedantigen binding protein comprising a variable light chain comprising SEQID NO. 30 (CAN-SSMQC23-VL) and a variable heavy chain comprising SEQ IDNO. 27 (CAN-SSM57-VH).

In one or more embodiments, the present invention provides a veterinarycomposition comprising a therapeutically effective amount of any one ormore of the isolated antigen binding proteins of the present invention.

In one or more embodiment the veterinary composition of the inventionhas no significant adverse effect on the immune system of a canine.

In one or more embodiments, the present invention provides a host cellthat produces any one or more of the antigen binding proteins of thepresent invention.

In one embodiment, the invention provides an isolated nucleic acid thatcomprises a nucleic acid sequence encoding a caninized antigen bindingprotein selected from the group consisting of:

nucleic acids encoding a variable heavy chain (VH) selected from thegroup consisting of: SEQ ID NO. 32 (MU-RN911-VHnt); SEQ ID NO: 34(CAN-N2G9-VHnt); SEQ ID NO: 38 (CAN-LTM109-VHnt); SEQ ID NO: 40(CAN-SSM57-VHnt); SEQ ID NO: 41 (CAN-SSM58-VHnt); SEQ ID NO: 42(CAN-SSM66-VHnt); and variants thereof having one or more conservativenucleic acid substitutions.

In one or more embodiments, the invention provides an isolated nucleicacid that comprises a nucleic acid sequence encoding a caninized antigenbinding protein selected from the group consisting of:

nucleic acids encoding a variable light chain (VL) selected from thegroup consisting of: SEQ ID NO. 31 (MU-RN911-VLnt); SEQ ID NO: 33(CAN-E3M-VLnt) ; SEQ ID NO: 35 (CAN-618-VLnt); SEQ ID NO: 36(CAN-QC23-VLnt) ; SEQ ID NO: 37 (CAN-LTM109-VLnt); SEQ ID NO: 39(CAN-SSME3M-VLnt); SEQ ID NO: 43 (CAN-SSMQC23-VLnt); and variantsthereof having one or more conservative nucleic acid substitutions.

In some embodiments the invention further comprises nucleic acidsencoding a variable heavy chain (VH) selected from the group consistingof: SEQ ID NO. 32 (MU-RN911-VHnt); SEQ ID NO: 34 (CAN-N2G9-VHnt); SEQ IDNO: 38 (CAN-LTM109-VHnt); SEQ ID NO: 40 (CAN-SSM57-VHnt); SEQ ID NO: 41(CAN-SSM58-VHnt); or SEQ ID NO: 42 (CAN-SSM66-VHnt); and variantsthereof having one or more conservative nucleic acid substitutions.

In one or more embodiments, the present invention provides isolatednucleic acid wherein the nucleic acid encodes the variable light chainof a caninized anti-NGF antigen binding protein which comprises SEQ IDNO. 33 (CAN-E3M-VLnt) and the nucleic acid encoding the variable heavychain which comprises SEQ ID NO. 34 (CAN-N2G9-VHnt).

In one or more embodiments, the present invention provides isolatednucleic acid wherein the nucleic acid encodes the variable light chainwhich comprises SEQ ID NO: 39 (CAN-SSME3M-VLnt) and the nucleic acidencoding the variable heavy chain which comprises SEQ ID NO: 40(CAN-SSM57-VHnt).

In one or more embodiments, the present invention provides isolatednucleic acid wherein the nucleic acid encodes the variable light chainwhich comprises SEQ ID SEQ ID NO: 43 (CAN-SSMQC23-VLnt) and the nucleicacid encoding the variable heavy chain which comprises SEQ ID NO: 40(CAN-SSM57-VHnt).

In one or more embodiments, the invention provides a vector comprisingthe any one or more of the nucleic acids of the present invention.

In one or more embodiments, the invention provides a host cellcomprising the any one or more of the nucleic acids of the presentinvention.

In one or more embodiments, the invention provides a host cellcomprising the vector that comprises any one or more of the nucleicacids of the present invention.

In one embodiment, the invention provides a method of producing anantigen binding protein comprising culturing any of the host cells ofthe present invention as described herein, under conditions that resultin production of the caninized antigen binding protein, and isolatingthe caninized antibody antigen binding protein from the host cell orculture medium of the host cell.

In one embodiment, the present invention provides a method of treating acanine for an NGF related disorder comprising administering atherapeutically effective amount of the veterinary composition of theinvention. In some embodiments, the NGF related disorder is selectedfrom the group consisting of: cardiovascular diseases, atherosclerosis,obesity, diabetes, metabolic syndrome, pain and inflammation. In someembodiments of the present invention the type of pain is selected fromthe group consisting of: chronic pain; inflammatory pain, post-operativeincision pain, neuropathic pain, fracture pain, osteoporotic fracturepain, post-herpetic neuralgia, cancer pain, pain resulting from burns,pain associated with wounds, pain associated with trauma, neuropathicpain, pain associated with musculoskeletal disorders such as rheumatoidarthritis, osteoarthritis, ankylosing spondylitis, seronegative(non-rheumatoid) arthropathies, non-articular rheumatism andperiarticular disorders and peripheral neuropathy. In some embodiments,the type of pain is osteoarthritis pain.

In one or more embodiments, the present invention provides a method ofinhibiting NGF activity in a canine by administering the veterinarycomposition of the present invention.

In one embodiment, the present invention provides a method of detectingor quantitating NGF levels in a biological sample, the methodcomprising:

-   -   (a) incubating a clinical or biological sample containing NGF in        the presence of any one of the caninized antibody, antigen        binding protein or fragments of the present invention; and    -   (b) detecting the antigen binding protein or fragments which are        bound to NGF in the sample.

In some embodiments the antigen binding protein or fragments isdetectably labeled. In some embodiments the antigen binding protein orfragments is unlabelled is used in combination with a second antigenbinding protein or fragments which is detectably labeled. In oneembodiment the invention comprises a kit comprising the antigen bindingprotein of the invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: is a schematic representation of the general structure of amouse immunoglobulin G (IgG) molecule highlighting the antigen bindingsite.

FIG. 2: is a schematic representation of the general structure of amouse/canine chimeric IgG.

FIG. 3: is an illustration showing speciation or “caninization” of amouse IgG, mouse CDRs are grafted onto canine frameworks.

FIG. 4 is an illustration of a “heterochimeric monoclonal antibodyparing the chimeric light chain with a fully caninized heavy chain.

FIG. 5 is an illustration of antibody variable chains showing primers toconstant regions and degenerate primers directed at mouse variableregions.

FIG. 6. (A) Molecular modeling of the RN 911 epitope on mature humanB-NGF. NGF typically is homodimerized. Each monomer of NGF isdifferentiated by the light and medium grey designations, and residuesidentified that affect RN911 binding are shown in black. Each of thevariable regions that impact binding of the various murine mAbs islabeled accordingly. (B) Amino acid sequence conservation across speciesof the RN911 binding epitope on B-NGF. Amino acids critical for RN911binding to NGF are shaded in gray. SEQ ID NOs 52-54 describing the aminoacid sequences of dog, cat, human, mouse and rat NGF are noted.

FIG. 7 is a graphical representation of serum levels of free RN911 afterIV administration to beagle dogs.

FIG. 8 is a graphical representation of serum levels of “bound NGF”(i.e., NGF bound to RN911) after IV administration to beagle dogs.

FIG. 9: is a graphical representation of the effect of RN911 (3 mg/kg,IV) on lameness in a beagle (LPS-induced) synovitis pain model. *P<0.1as compared with PBS vehicle.

FIG. 10 is a schematic representation of an example of frameworksubstitutions made to light chains to regain expression and retainbinding. SEQ ID NOs 57-61 describing the sequences are noted

FIG. 11 summarizes the results of the expression and the mutations ofthe caninized derivatives using different framework sequences.

FIG. 12A and B is a graphical representation of affinity data ofcaninized mAb (left). (Right) trkA inhibition data with pre-incubatedmAb+NGF. (Bottom)

FIG. 13 is a graphical representation of affinity data of the caninizedmAb determined by cell-based assay using trkA expressing cell line todetermine EC50 of each mAb.

FIG. 14 is a graphical representation of the effects of caninizedAnti-NGF mAb PF 06442590 (CANSSM57-VH/CAN SSME3M-VL)

FIG. 15 is an illustration of the library design used forLook-Through-Mutagenesis (LTM) and Site-saturation mutagenesis (SSM)

FIG. 16 is a graphical representation of a pharmacokinetics (PK) studyof anti-NGF monoclonal antibodies in dogs as measured for thirty-fivedays.

FIG. 17 is a schematic depiction of inflammation induction byintra-articular injection of MIA used to reproduce OA-like lesions inrat (MIA rat pain model).

FIGS. 18 and 19 is a graphical representation of results after paintreatment (morphine, monoclonal antibodies) in the MIA rat pain model

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence for the variable heavy chainCDR1 referred to herein as RN911-VH-CDR1

SEQ ID NO: 2 is the amino acid sequence for the variable heavy chainCDR2 referred to herein as RN911-VH-CDR2

SEQ ID NO: 3 is the amino acid sequence for the variable heavy chainCDR2 referred to herein as RN911-VH-CDR3

SEQ ID NO: 4 is the amino acid sequence for the variable light chain CDR1 referred to herein as RN911-VL-CDR1

SEQ ID NO: 5 is the amino acid sequence for the variable light chainCDR2 referred to herein as RN911-VL-CDR2

SEQ ID NO: 6 is the amino acid sequence for the variable light chainCDR3 referred to herein as RN911-VL-CDR3

SEQ ID NO: 7 is the amino acid sequence for the variable heavy chainCDR1 referred to herein as SSM57-VH-CDR1

SEQ ID NO: 8 is the amino acid sequence for the variable heavy chainCDR1 referred to herein as SSM58-VH-CDR1

SEQ ID NO: 9 is the amino acid sequence for the variable heavy chainCDR3 referred to herein as LTM109-VH-CDR3 and SSM57-VH CDR3

SEQ ID NO: 10 is the amino acid sequence for the variable light chainCDR1 referred to herein as LTM109-VL-CDR1

SEQ ID NO: 11 is the amino acid sequence for the variable light chainCDR2 referred to herein as SSM57-VL-CDR2

SEQ ID NO: 12 is the amino acid sequence for the variable light chainCDR3 referred to herein as LTM109-VL-CDR3

SEQ ID NO: 13 is the amino acid sequence for the variable light chainCDR2 referred to herein as SSM66-VH-CDR2 and SSM57-VH CDR2

SEQ ID NO: 14 is the amino acid sequence for the variable heavy chainreferred to herein as MU-RN911-VH

SEQ ID NO: 15 is the amino acid sequence for the variable light chainreferred to herein as MU-RN911-VL

SEQ ID NO: 16 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-E3M-VL

SEQ ID NO: 17 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-N2G9-VH

SEQ ID NO: 18 is the amino acid sequence for the variable light chainreferred to herein as CAN-618-VL

SEQ ID NO: 19 is the amino acid sequence for the variable light chainreferred to herein as CAN-QC23-VL

SEQ ID NO: 20 is the amino acid sequence for the variable light chainreferred to herein as CAN-618FW1-VL

SEQ ID NO: 21 is the amino acid sequence for the variable light chainreferred to herein as CAN-618FW2-VL

SEQ ID NO: 22 is the amino acid sequence for the variable light chainreferred to herein as CAN-618FW3-VL

SEQ ID NO: 23 is the amino acid sequence for the variable light chainreferred to herein as CAN-618FW4-VL

SEQ ID NO: 24 is the amino acid sequence for the variable light chainreferred to herein as CAN-LTM109-VL

SEQ ID NO: 25 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-LTM109-VH)

SEQ ID NO: 26 is the amino acid sequence for the variable light chainreferred to herein as CAN-SSME3M-VL)

SEQ ID NO: 27 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-SSM57-VH

SEQ ID NO: 28 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-SSM58-VH

SEQ ID NO: 29 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-SSM66-VH

SEQ ID NO: 30 is the amino acid sequence for the variable heavy chainreferred to herein as CAN-SSMQC23-VL

SEQ ID NO: 31 is the nucleotide sequence encoding the variable lightchain referred to herein as MU-RN911-VLnt

SEQ ID NO: 32 is the nucleotide sequence encoding the variable heavychain referred to herein as MU-RN911-VHnt)

SEQ ID NO: 33 is the nucleotide sequence encoding the variable lightchain referred to herein as CAN-E3M-VLnt

SEQ ID NO: 34 is the nucleotide sequence encoding the variable heavychain referred to herein as CAN-N2G9-VHnt

SEQ ID NO: 35 is the nucleotide sequence encoding the variable lightchain referred to herein as CAN-618-VLnt

SEQ ID NO: 36 is the nucleotide sequence encoding the variable lightchain referred to herein as CAN-QC23-VLnt

SEQ ID NO: 37 is the nucleotide sequence encoding the variable lightchain referred to herein as CAN-LTM109-VLnt

SEQ ID NO: 38 is the nucleotide sequence encoding the variable heavychain referred to herein as CAN-LTM109-VHnt

SEQ ID NO: 39 is the nucleotide sequence encoding the variable lightchain referred to herein as CAN-SSME3M-VLnt

SEQ ID NO: 40 is the nucleotide sequence encoding the variable heavychain referred to herein as CAN-SSM57-VHnt

SEQ ID NO: 41 is the nucleotide sequence encoding the variable heavychain referred to herein as CAN-SSM58-VHnt

SEQ ID NO: 42 is the nucleotide sequence encoding the variable heavychain referred to herein as CAN-SSM66-VHnt

SEQ ID NO: 43 is the nucleotide sequence encoding the variable lightchain referred to herein as CAN-SSMQC23-VLnt

SEQ ID NO: 44 is the amino acid sequence for the canine heavy chainconstant region referred to herein as CAN-65E-HC

SEQ ID NO: 45 is the nucleotide sequence encoding the canine heavy chainconstant region referred to herein as CAN-65E-HCnt)

SEQ ID NO: 46 is the amino acid sequence for the canine kappa constantlight chain region and referred to herein as CAN-KAPPA-LC

SEQ ID NO: 47 is the nucleotide sequence for the canine kappa constantlight chain region and referred to herein as CAN-KAPPA-LCnt

SEQ ID NO: 48 is the nucleotide sequence for the canine heavy chainconstant region referred to herein as CAN-65 HCnt

SEQ ID NO. 49 is the amino acid sequence for the canine heavy chainconstant region referred to herein as CAN-65 HC

SEQ ID NO. 50 is the amino acid sequence of Canis lupus familiaris NerveGrowth Factor Genbank Accession No. AAY16195

SEQ ID NO. 51 is the nucleotide sequence of Canis lupus familiaris NerveGrowth Factor

SEQ ID NO. 52 is the amino acid sequence of Canine Nerve Growth Factor,partial sequence as seen in FIG. 6B

SEQ ID NO. 53 is the amino acid sequence of Feline Nerve Growth Factor,partial sequence as seen in FIG. 6B

SEQ ID NO. 54 is the amino acid sequence of Human Nerve Growth Factor,partial sequence as seen in FIG. 6B

SEQ ID NO. 55 is the amino acid sequence of Murine Nerve Growth Factor,partial sequence as seen in FIG. 6B

SEQ ID NO. 56 is the amino acid sequence of Rat Nerve Growth Factor,partial sequence as seen in FIG. 6B

SEQ ID NO. 57 is the amino acid sequence of framework substitutions madeto light chain variable region to regain expression and retain bindingof RN911.

SEQ ID NO. 58 is the amino acid sequence of framework substitutions madeto light chain variable region to regain expression and retain bindingof Can911_gapped.

SEQ ID NO. 59 is the amino acid sequence of framework substitutions madeto light chain variable region to regain expression and retain bindingCan911.

SEQ ID NO. 60 is the amino acid sequence of framework substitutions madeto light chain variable region to regain expression and retain bindingof QC23LC

SEQ ID NO. 61 is the consensus amino acid sequence of the light chainvariable region after framework substitutions of SEQ ID NOs 57-60.

SEQ ID NO.62 is the amino acid sequence of the RN911heavy chain variableregion

SEQ ID NO.63 is the amino acid sequence of the E3 heavy chain variableregion

SEQ ID NO.64 is the amino acid sequence of the A01 heavy chain variableregion

SEQ ID NO.65 is the amino acid sequence of the B01 heavy chain variableregion

SEQ ID NO.66 is the amino acid sequence of the B06 heavy chain variableregion

SEQ ID NO.67 is the amino acid sequence of the F03 heavy chain variableregion

SEQ ID NO.68 is the amino acid sequence of the F06 heavy chain variableregion

SEQ ID NO.69 is the amino acid sequence of the D10 heavy chain variableregion

SEQ ID NO.70 is the amino acid sequence of the E07 heavy chain variableregion

SEQ ID NO.71 is the amino acid sequence of the C07 heavy chain variableregion

SEQ ID NO.72 is the amino acid sequence of the C09 heavy chain variableregion

SEQ ID NO.73 is the amino acid sequence of the A02 heavy chain variableregion

SEQ ID NO.74 is the amino acid sequence of the D08 heavy chain variableregion

SEQ ID NO.75 is the amino acid sequence of the D09 heavy chain variableregion

SEQ ID NO.76 is the amino acid sequence of the All heavy chain variableregion

SEQ ID NO.77 is the amino acid sequence of the C12 heavy chain variableregion

SEQ ID NO.78 is the amino acid sequence of the H11 heavy chain variableregion

SEQ ID NO.79 is the amino acid sequence of the F11 heavy chain variableregion

SEQ ID NO.80 is the amino acid sequence of the A08 heavy chain variableregion

SEQ ID NO.81 is the amino acid sequence of the C06 heavy chain variableregion

SEQ ID NO.82 is the amino acid sequence of the ImP2_E06 heavy chainvariable region

SEQ ID NO.83 is the amino acid sequence of the ImP2_C08 heavy chainvariable region

SEQ ID NO.84 is the amino acid sequence of the ImP2_H06 heavy chainvariable region

SEQ ID NO.85 is the amino acid sequence of the ImP1_G12 heavy chainvariable region

SEQ ID NO.86 is the amino acid sequence of the ImP1_B02 heavy chainvariable region

SEQ ID NO.87 is the amino acid sequence of the ImP2_G12 heavy chainvariable region

SEQ ID NO.88 is the amino acid sequence of the ImP2_G02 heavy chainvariable region

SEQ ID NO.89 is the amino acid sequence of the ImmRB1lheavy chainvariable region

SEQ ID NO. 90 is the amino acid sequence of the ImmRD07 heavy chainvariable region

SEQ ID NO.91 is the amino acid sequence of the ImmRC08 heavy chainvariable region

SEQ ID NO.92 is the amino acid sequence of the ImmRE08 heavy chainvariable region

SEQ ID NO.93 is the amino acid sequence of the ImmRA06 heavy chainvariable region

SEQ ID NO.94 is the amino acid sequence of the ImmRA10 heavy chainvariable region

SEQ ID NO.95 is the amino acid sequence of the ImmRH02 heavy chainvariable region

SEQ ID NO.96 is the amino acid sequence of the ImmRG09 heavy chainvariable region

SEQ ID NO.97 is the amino acid sequence of the ImmRD12 heavy chainvariable region

SEQ ID NO.98 is the amino acid sequence of the ImmRE04 heavy chainvariable region

SEQ ID NO.99 is the amino acid sequence of the ImmRC06 heavy chainvariable region

SEQ ID NO.100 is the amino acid sequence of the ImmRG02 heavy chainvariable region

SEQ ID NO.101 is the amino acid sequence of the ImmRA11 heavy chainvariable region

SEQ ID NO.102 is the amino acid sequence of the ImmRC02 heavy chainvariable region

SEQ ID NO.103 is the amino acid sequence of the ImmRG03 heavy chainvariable region

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein provides caninized anti-NGF antigenbinding proteins that bind canine NGF with high affinity. The inventionfurther provides caninized antigen binding proteins and polypeptidesthat also bind to canine NGF that are variants of said antigen bindingproteins as well as methods of making and using these antigen bindingproteins. In some embodiments, the invention also providespolynucleotides encoding said antigen binding proteins and/orpolypeptide. The invention disclosed herein also provides methods forpreventing and/or treating pain in a canine by administration of atherapeutically effective amount of the caninized anti-NGF antigenbinding proteins.

General Techniques

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such may vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

Unless otherwise defined, scientific and technical terms used inconnection with the antibodies described herein shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application

Standard techniques are used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transfection (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures are generally performed according to conventional methodswell known in the art and as described, but not limited to the variousgeneral and more specific references that are cited and discussedthroughout the present specification, See e.g., Sambrook et al.MOLECULAR CLONING: LAB. MANUAL (3^(rd) ed., Cold Spring Harbor Lab.Press, Cold Spring Harbor, N.Y., 2001) and Ausubel et al. CurrentProtocols in Molecular Biology (New York: Greene Publishing AssociationJ Wiley Interscience), Oligonucleotide Synthesis (M. J. Gait, ed.,1984);Methods in Molecular Biology, Humana Press; Cell Biology: A LaboratoryNotebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture(R. l. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (l.P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and TissueCulture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G.Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology(Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weirand C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987); PCR: ThePolymerase Chain Reaction, (Mullis et al., eds., 1994); CurrentProtocols in Immunology (E. Coligan et al., eds., 1991); Short Protocolsin Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies:a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonalantibodies: a practical approach (P. Shepherd and C. Dean, eds., OxfordUniversity Press, 2000); Using antibodies: a laboratory manual (E.Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); TheAntibodies (M. Zanetti and J. D. Capra, eds., Harwood AcademicPublishers, 1995); and Cancer: Principles and Practice of Oncology (Y.T. DeVita et al., eds., J.B. Lippincott Company, 1993).

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.”

Definitions

Before describing the present invention in detail, several terms used inthe context of the present invention will be defined. In addition tothese terms, others are defined elsewhere in the specification asnecessary. Unless otherwise expressly defined herein, terms of art usedin this specification will have their art-recognized meanings.

As used in the specification and claims, the singular form “a”, “an” and“the” includes plural references unless the context clearly dictatesotherwise. For example, reference to “an antibody” includes a pluralityof such antibodies.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers.

As used herein, the term “nerve growth factor” and “NGF” refers to nervegrowth factor and variants thereof that retain at least part of thebiological activity of NGF.

“NGF receptor” refers to a polypeptide that is bound by or activated byNGF. NGF receptors include the TrkA receptor and to a lesser extent thep75 receptor of canines.

“Biological activity” of NGF generally refers to the ability to bind NGFreceptors and/or activate NGF receptor signaling pathways. Withoutlimitation, a biological activity includes anyone or more of thefollowing: the ability to bind an NGF receptor (such as TrkA and/orp75); the ability to promote TrkA receptor dimerization and/orautophosphorylation; the ability to activate an NGF receptor signalingpathway; the ability to promote cell differentiation, proliferation,survival, growth and other changes in cell physiology, including (in thecase of neurons, including peripheral and central neuron) change inneuronal morphology, synaptogenesis, synaptic function, neurotransmitterand/or neuropeptide release and regeneration following damage; theability to promote survival of mouse E13.5 trigeminal neurons; and theability to mediate pain, including post-surgical pain.

As used herein, an “anti-NGF antigen binding protein” (interchangeablytermed “anti-NGF antibody” and “anti-NGF antagonist antibody”) refers toan antigen binding protein which is able to bind to NGF and inhibit NGFbiological activity and/or downstream pathway(s) mediated by NGFsignaling. An anti-NGF antagonist antibody encompasses antibodies thatblock, antagonize, suppress or reduce (including significantly) NGFbiological activity, including downstream pathways mediated by NGFsignaling and/or inhibit NGF from binding to its receptor trkA, such asreceptor binding and/or elicitation of a cellular response to NGF. Forpurpose of the present invention, it will be explicitly understood thatthe term “anti-NGF antagonist antibody” encompass all the previouslyidentified terms, titles, and functional states and characteristicswhereby the NGF itself, an NGF biological activity (including but notlimited to its ability to ability to mediate any aspect of post-surgicalpain), or the consequences of the biological activity, are substantiallynullified, decreased, or neutralized in any meaningful degree. In someembodiments, an anti-NGF antagonist antibody binds NGF and prevent NGFdimerization and/or binding to an NGF receptor (such as TrkA and/orp75). In other embodiments, an anti-NGF antibody binds NGF and preventsTrkA receptor dimerization and/or TrkA autophosphorylation. Examples ofanti-NGF antagonist antibodies are provided herein.

As used herein, the term “antigen binding protein”, “antibody” and thelike, which may be used interchangeably, refers to a polypeptide, orfragment thereof, comprising an antigen binding site. In one embodimentof the present invention the antigen binding protein of the inventionfurther provides an intact immunoglobulin capable of specific binding toa target, such as a carbohydrate, polynucleotide, lipid, polypeptide,etc., through at least one antigen recognition site located in thevariable region of the immunoglobulin molecule. An intact antibody hastwo light and two heavy chains. Thus a single isolated intact antibodymay be a polyclonal antibody, a monoclonal antibody, a syntheticantibody, a recombinant antibody, a chimeric antibody, a heterochimericantibody. The term “antigen binding protein” “antibody’ preferablyrefers to monoclonal antibodies and fragments thereof, and immunologicbinding equivalents thereof that can bind to the NGF protein andfragments thereof. The term antibody and antigen binding protein areused to refer to a homogeneous molecular, or a mixture such as a serumproduct made up of a plurality of different molecular entities. As usedherein, the term encompasses not only intact polyclonal or monoclonalantibodies, but also fragments thereof. For the purposes of the presentinvention, “antibody” and “antigen binding protein” also includesantibody fragments, unless otherwise stated. Exemplary antibodyfragments include Fab, Fab′, F(ab′)2, Fv, scFv, Fd, dAb, diabodies,their antigen-recognizing fragments, small modular immunopharmaceuticals(SMIPs) nanobodies, IgNAR molecules and the like all recognized by oneof skill in the art to be an antigen binding protein or antibodyfragment and any of above mentioned fragments and their chemically orgenetically manipulated counterparts, as well as other antibodyfragments and mutants thereof, fusion proteins comprising an antibodyportion, and any other modified configuration of the immunoglobulinmolecule that comprises an antigen recognition site. Antibodies andantigen binding proteins can be made, for example, via traditionalhybridoma techniques (Kohler et al., Nature 256:495-499 (1975)),recombinant DNA methods (U.S. Pat. No. 4,816,567), or phage displaytechniques using antibody libraries (Clackson et al., Nature 352:624-628(1991); Marks et al., J. Mol. Biol. 222:581-597 (1991)). For variousother antibody production techniques, see Antibodies: A LaboratoryManual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988 as wellas other techniques that are well known to those skilled in the art.

A “monoclonal antibody” as defined herein is an antibody produced by asingle clone of cells (specifically, a single clone of hybridoma cells)and therefore a single pure homogeneous type of antibody. All monoclonalantibodies produced from the same clone are identical and have the sameantigen specificity. Monoclonal antibodies are a homogeneous antibodypopulation wherein the monoclonal antibody is comprised of amino acids(naturally occurring and non-naturally occurring) that are involved inthe selective binding of an antigen. A population of monoclonalantibodies is highly specific, being directed against a single antigenicsite. The term “monoclonal antibody” encompasses not only intactmonoclonal antibodies and full-length monoclonal antibodies, but alsofragments thereof (Fab, Fab′, F(ab′)2, Fv, scFv, Fd, dAb, diabodies,their antigen-recognizing fragments, small modular immunopharmaceuticals(SMIPs) nanobodies, IgNAR molecules and the like), mutants thereof,fusion proteins comprising an antibody portion, and any other modifiedconfiguration of the immunoglobulin molecule that comprises an antigenrecognition site of the required specificity and the ability to bind toan antigen. It is not intended to be limited as regards to the source ofthe antibody or the manner in which it is made (e.g., by hybridoma,phage selection, recombinant expression, transgenic animals, etc.).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species, while the remainder ofthe chain(s) is identical with or homologous to corresponding sequencesin antibodies derived from another species, as well as fragments of suchantibodies, so long as they exhibit the desired biological activity.Typically, chimeric antibodies are antibodies whose light and heavychain genes have been constructed, typically by genetic engineering,from antibody variable and constant region genes belonging to differentspecies. For example, the variable segments of the genes from a mousemonoclonal antibody may be joined to canine constant segments. FIG. 2 isa schematic representation of the general structure of one embodiment ofa mouse: canine IgG. In this embodiment, the antigen binding site isderived from mouse while the Fc portion is canine.

The term “heterochimeric” as defined herein, refers to an antibody inwhich one of the antibody chains (heavy or light) is caninized while theother is chimeric. FIG. 4 depicts one embodiment of a heterochimericmolecule. In this embodiment, a caninized variable heavy chain (whereall of the CDRs are mouse and all FRs are canine) is paired with achimeric variable light chain (where all of the CDRs are mouse and allFRs are mouse. In this embodiment, both the variable heavy and variablelight chains are fused to a canine constant region.

“Caninized” forms of non-canine (e.g., murine) antibodies aregenetically engineered antibodies that contain minimal sequence derivedfrom non-canine immunoglobulin. “Caninization” is defined as a methodfor transferring non-canine antigen-binding information from a donorantibody to a less immunogenic canine antibody acceptor to generatetreatments useful as therapeutics in dogs. Caninized antibodies arecanine immunoglobulins (recipient antibody) in which hypervariableregion residues of the recipient are replaced by hypervariable regionresidues from a non-canine species (donor antibody) such as such asmouse, rat, rabbit, cat, dogs, goat, chicken, bovine, horse, llama,camel, dromedaries, sharks, non-human primates, human, humanized,recombinant sequence, or an engineered sequence having the desiredproperties , specificity, affinity, and capacity. In some instances,framework region (FR) residues of the canine immunoglobulin are replacedby corresponding non-canine residues. Furthermore, caninized antibodiesmay include residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance. The modifications to the hypervariable regionsand/or the framework regions, as described herein, are determined foreach separately engineered speciated (caninized) antibody based onexperimentation known to those in the art and cannot be predicted priorto said experimentation. In general, the caninized antibody will includesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable regionscorrespond to those of a non-canine immunoglobulin and all orsubstantially all of the FRs are those of a canine immunoglobulinsequence. The caninized antibody optionally also will comprise acomplete, or at least a portion of an immunoglobulin constant region(Fc), typically that of a canine immunoglobulin. FIG. 3 is anillustration of one embodiment showing speciation or caninization of amouse IgG. In this embodiment, mouse CDRs are grafted onto canineframeworks. In some cases, mouse frameworks or residues therein that areoutside of the hypervariable region are maintained.

The phrase “recombinant canine antibody” includes canine antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial canine antibody library, antibodies isolated from ananimal (e.g., a mouse) that is transgenic for canine immunoglobulingenes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res.20:6287-6295) or antibodies prepared, expressed, created or isolated byany other means that involves splicing of canine immunoglobulin genesequences to other DNA sequences.

The term “canine antibody”, as used herein, refers to a canine antibodythat is generated against a target and is prepared by hybridoma methodswell known to one skilled in the art and described herein.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 Daltons, composed of twoidentical light (l) chains and two identical heavy (H) chains. Eachlight chain is linked to a heavy chain by one covalent disulfide bond,while the number of disulfide linkages varies among the heavy chains ofdifferent immunoglobulin isotypes. Each heavy and light chain also hasregularly spaced intrachain disulfide bridges. Each heavy chain has atone end a variable domain (VH) followed by a number of constant domains.Each light chain has a variable domain at one end (VL) and a constantdomain at its other end; the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and thelight-chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light- and heavy-chain variable domains. FIG. 1 isan example of the general structure of a native mouse immunoglobulin G(IgG) highlighting the antigen binding site.

The “parent” antibody herein is one that is encoded by an amino acidsequence used for the preparation of the variant. Preferably, the parentantibody has a canine framework region and, if present, has canineantibody constant region(s). For example, the parent antibody may be acaninized or canine antibody.

Depending on the amino acid sequence of the constant domain of the heavychains of antibodies, immunoglobulins can be assigned to differentclasses. Presently there are five major classes of immunoglobulins: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA, and IgA₂ (asdefined by mouse and human designation). The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known in multiple species. The prevalence ofindividual isotypes and functional activities associated with theseconstant domains are species-specific and must be experimentallydefined.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (K) and lambda (λ), based on the amino acid sequences of theirconstant domains.

A “variable region” of an antibody refers to the variable region of theantibody light chain or the variable region of the antibody heavy chain,either alone or in combination. The variable regions of the heavy andlight chain each consist of four framework regions (FR) connected bythree complementarity determining regions (CDRs) also known ashypervariable regions. The CDRs in each chain are held together in closeproximity by the FRs and, with the CDRs from the other chain, contributeto the formation of the antigen-binding site of antibodies. There are atleast two techniques for determining CDRs: (I) an approach based oncross-species sequence variability (i.e., Kabat et al. Sequences ofProteins of Immunological Interest, (5th ed., 1991, National Institutesof Health, Bethesda Md.)); and (2) an approach based on crystallographicstudies of antigen-antibody complexes (Chothia et al. (1989) Nature342:877; AI-Iazikani et al (1997) J. Molec. Bioi. 273:927-948)). As usedherein, a CDR may refer to CDRs defined by either approach or by acombination of both approaches.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen binding.The hypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (Kabat, et al. (1991),above) and/or those residues from a “hypervariable loop” (Chothia andLesk J. Mol. Biol. 196:901-917 (1987). “Framework” or “FR” residues arethose variable domain residues other than the hypervariable regionresidues as herein defined.

As used herein, the term “antigen binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody binding region includes the“framework” amino acid residues necessary to maintain the properconformation of the antigen-binding residues.

A “functional Fc region” possesses at least one effector function of anative sequence Fc region. Exemplary “effector functions” include C1qbinding; complement dependent cytotoxicity (CDC); Fc receptor binding;neonatal receptor binding; antibody-dependent cell-mediated cytotoxicity(ADCC); phagocytosis; down-regulation of cell surface receptors (e.g. Bcell receptor; BCR), etc. Such effector functions generally require theFc region to be combined with a binding domain (e.g. an antibodyvariable domain) and can be assessed using various assays known in theart for evaluating such antibody effector functions.

A “native sequence Fc region” comprises an amino acid sequence identicalto the amino acid sequence of an Fc region found in nature. A “variantFc region” or a “mutated” or “mutant” Fc region comprises an amino acidsequence which differs from that of a native sequence Fc region byvirtue of at least one amino acid modification, and may or may notretain at least one effector function of the native sequence Fc region.Preferably, the variant Fc region has at least one amino acidsubstitution compared to a native sequence Fc region or to the Fc regionof a parent polypeptide, e.g. from about one to about ten amino acidsubstitutions, and preferably from about one to about five amino acidsubstitutions in a native sequence Fc region or in the Fc region of theparent polypeptide. The variant Fc region herein will preferably possessat least about 80% sequence identity with a native sequence Fc regionand/or with an Fc region of a parent polypeptide, and most preferably atleast about 90% sequence identity therewith, more preferably at leastabout 95% sequence identity therewith. A variant or mutated Fc regionmay also essentially eliminate the function of the Fc region of theantibody. For example Fc region mutations may eliminate effectorfunction of the antibody. In one embodiment of the invention theantibody of the invention comprises a mutated Fc region.

As used herein, “Fc receptor” and “FcR” describe a receptor that bindsto the Fc region of an antibody. The preferred FcR is a native sequenceFcR. Moreover, a preferred FcR is one which binds an IgG antibody (agamma receptor) and includes receptors of the FcyRI, FcyRII, and FcyRIIIsubclasses, including allelic variants and alternatively spliced formsof these receptors. FcyRII receptors include FcyRIIA (an “activatingreceptor”) and FcyRIIB (an “inhibiting receptor”), which have similaramino acid sequences that differ primarily in the cytoplasmic domainsthereof. FcRs are reviewed in Ravetch and Kinet, 1991, Ann. Rev.Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and deHaas et al., 1995, J. Lab. Clin. Med., 126:330-41. “FcR” also includesthe neonatal receptor, FcRn, which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., 1976, J. Immunol., 117:587;and Kim et al., 1994, J. Immunol., 24:249).

As used herein “antibody-dependent cell-mediated cytotoxicity” and“ADCC” refer to a cell-mediated reaction in which nonspecific cytotoxiccells that express Fc receptors (FcRs) (e.g. natural killer (NK) cells,neutrophils, and macrophages) recognize bound antibody on a target celland subsequently cause lysis of the target cell. ADCC activity of amolecule of interest can be assessed using an in vitro ADCC assay, suchas that described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and NK cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., 1998, PNAS (USA),95:652-656.

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget in the presence of complement. The complement activation pathwayis initiated by the binding of the first component of the complementsystem (C1q) to a molecule (e.g. an antibody) complexed with a cognateantigen. To assess complement activation, a CDC assay, e.g. as describedin Gazzano-Santoro et al., J. Immunol. Methods, 202: 163 (1996), may beperformed.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxyl terminus of the heavy chain CH1 domain including one or morecysteine(s) from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′) 2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

“Fv’ is the minimum antibody fragment that contains a completeantigen-recognition and binding site. This region consists of a dimer ofone heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

An “antigen”, as used herein, refers to the antigenic determinantrecognized by the CDRs of the antigen binding protein or antibody asdescribed herein. In other words, epitope refers to that portion of anymolecule capable of being recognized by, and bound by, an antibody.Unless indicated otherwise, the term “epitope” as used herein, refers tothe region of NGF to which an anti-NGF antigen bindingprotein/antibody/agent binds.

The term “antigen binding domain,” “active fragments of an antibody” orthe like refers to the part of an antibody or antigen binding proteinthat comprises the area specifically binding to or complementary to apart or all of an antigen. Where an antigen is large, an antibody mayonly bind to a particular part of the antigen. The “epitope,” “activefragments of an epitope,” or “antigenic determinant” or the like is aportion of an antigen molecule that is responsible for specificinteractions with the antigen binding domain of an antibody. An antigenbinding domain may be provided by one or more antibody variable domains(e.g., a so-called Fd antibody fragment consisting of a VH domain). Anantigen binding domain may comprise an antibody light chain variabledomain (VL) and an antibody heavy chain variable domain (VH) (U.S. Pat.No. 5,565,332).

The terms “binding portion” of an antibody (or “antibody portion”) orantigen-binding polypeptide or the like includes one or more completedomains, e.g., a pair of complete domains, as well as fragments of anantibody that retain the ability to specifically bind to an antigen,e.g., NGF. It has been shown that the binding function of an antibodycan be performed by fragments of a full-length antibody. Bindingfragments are produced by recombinant DNA techniques, or by enzymatic orchemical cleavage of intact immunoglobulins. Binding fragments includeFab, Fab′, F(ab′) 2, Fabc, Fd, dAb, Fv, single chains, single-chainantibodies, e.g., scFv, and single domain antibodies (Muyldermans etal., 2001, 26:230-5), and an isolated complementarity determining region(CDR). Fab fragment is a monovalent fragment consisting of the VL, VH,CL and CH1 domains. F(ab)₂ fragment is a bivalent fragment comprisingtwo Fab fragments linked by a disulfide bridge at the hinge region. Fdfragment consists of the VH and CH1 domains, and Fv fragment consists ofthe VL and VH domains of a single arm of an antibody. A dAb fragmentconsists of a VH domain (Ward et al., (1989) Nature 341:544-546). Whilethe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv) (Bird et al., 1988, Science 242:423-426). Such singlechain antibodies are also intended to be encompassed within the term“binding portion” of an antibody. Other forms of single chainantibodies, such as diabodies are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see e.g., Holliger, et al.,1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). An antibody or bindingportion thereof also may be part of a larger immunoadhesion moleculesformed by covalent or non-covalent association of the antibody orantibody portion with one or more other proteins or peptides. Examplesof such immunoadhesion molecules include use of the streptavidin coreregion to make a tetrameric scFv molecule (Kipriyanov, S. M., et al.(1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteineresidue, a marker peptide and a C-terminal polyhistidine tag to makebivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al.(1994) Mol. Immunol. 31:1047-1058). Binding fragments such as Fab andF(ab′) 2 fragments, can be prepared from whole antibodies usingconventional techniques, such as papain or pepsin digestion,respectively, of whole antibodies. Moreover, antibodies, antibodyportions and immunoadhesion molecules can be obtained using standardrecombinant DNA techniques, as described herein and as known in the art.Other than “bispecific” or “bifunctional” antibodies, an antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites. Abispecific antibody can also include two antigen binding regions with anintervening constant region. Bispecific antibodies can be produced by avariety of methods including fusion of hybridomas or linking of Fab′fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol.79:315-321, 1990.; Kostelny et al., 1992, J. Immunol. 148, 1547-1553.

The term “backmutation” refers to a process in which some or all of thesomatically mutated amino acids of a canine antibody are replaced withthe corresponding germline residues from a homologous germline antibodysequence. The heavy and light chain sequences of the canine antibody ofthe invention are aligned separately with the germline sequences toidentify the sequences with the highest homology. Differences in thecanine antibody of the invention are returned to the germline sequenceby mutating defined nucleotide positions encoding such different aminoacid. The role of each amino acid thus identified as candidate forbackmutation should be investigated for a direct or indirect role inantigen binding and any amino acid found after mutation to affect anydesirable characteristic of the canine antibody should not be includedin the final canine antibody; as an example, activity enhancing aminoacids identified by the selective mutagenesis approach will not besubject to backmutation. To minimize the number of amino acids subjectto backmutation those amino acid positions found to be different fromthe closest germline sequence but identical to the corresponding aminoacid in a second germline sequence can remain, provided that the secondgermline sequence is identical and colinear to the sequence of thecanine antibody of the invention. Back mutation of selected targetframework residues to the corresponding donor residues might be requiredto restore and or improved affinity.

As used herein, “immunospecific” binding of antibodies refers to theantigen specific binding interaction that occurs between theantigen-combining site of an antibody and the specific antigenrecognized by that antibody (i.e., the antibody reacts with the proteinin an ELISA or other immunoassay, and does not react detectably withunrelated proteins). An epitope that “specifically binds”, or“preferentially binds” (used interchangeably herein) to an antibody or apolypeptide is a term well understood in the art, and methods todetermine such specific or preferential binding are also well known inthe art. A molecule is said to exhibit “specific binding” or“preferential binding” if it reacts or associates more frequently, morerapidly, with greater duration and/or with greater affinity with aparticular cell or substance than it does with alternative cells orsubstances. An antibody “specifically binds” or “preferentially binds”to a target if it binds with greater affinity, avidity, more readily,and/or with greater duration than it binds to other substances. Forexample, an antibody that specifically or preferentially binds to an NGFepitope is an antibody that binds this epitope with greater affinity,avidity, more readily, and/or with greater duration than it binds toother NGF epitopes or non-NGF epitopes. It is also understood by readingthis definition that, for example, an antibody (or moiety or epitope)that specifically or preferentially binds to a first target mayor maynot specifically or preferentially bind to a second target. As such,“specific binding” or “preferential binding” does not necessarilyrequire (although it can include) exclusive binding. Generally, but notnecessarily, reference to binding means preferential binding.

The term “specifically” in the context of antibody binding, refers tohigh avidity and/or high affinity binding of an antibody to a specificantigen, i.e., a polypeptide, or epitope. Antibody specifically bindingan antigen is stronger than binding of the same antibody to otherantigens. Antibodies which bind specifically to a polypeptide may becapable of binding other polypeptides at a weak, yet detectable level(e.g., 10% or less of the binding shown to the polypeptide of interest).Such weak binding, or background binding, is readily discernible fromthe specific antibody binding to a subject polypeptide, e.g. by use ofappropriate controls. In general, specific antibodies bind to an antigenwith a binding affinity with a K_(d) of 10⁻⁷ M or less, e.g., 10⁻⁸M orless e.g., 10⁻⁹ M or less, 10⁻¹⁰ or less, 10⁻¹¹ or less, 10⁻¹² or less,or 10⁻¹³ or less etc.

As used herein, the term “affinity” refers to the strength of thebinding of a single antigen-combining site with an antigenicdeterminant. Affinity depends on the closeness of stereochemical fitbetween antibody or antigen binding protein combining sites and antigendeterminants, on the size of the area of contact between them, on thedistribution of charged and hydrophobic groups, etc. Antibody affinitycan be measured by equilibrium analysis or by the Surface PlasmonResonance—“SPR” method (for example BIACORE™. The SPR method relies onthe phenomenon of surface plasmon resonance (SPR), which occurs whensurface plasmon waves are excited at a metal/liquid interface. Light isdirected at, and reflected from, the side of the surface not in contactwith sample, and SPR causes a reduction in the reflected light intensityat a specific combination of angle and wavelength. Bimolecular bindingevents cause changes in the refractive index at the surface layer, whichare detected as changes in the SPR signal.

The term “Kd’, as used herein, is intended to refer to the dissociationconstant of an antibody-antigen interaction. The dissociation constant,Kd, and the association constant, Ka, are quantitative measures ofaffinity. At equilibrium, free antigen (Ag) and free antibody (Ab) arein equilibrium with antigen-antibody complex (Ag−Ab), and the rateconstants, ka and kd, quantitate the rates of the individual reactions.At equilibrium, ka [Ab][Ag]=kd [Ag−Ab]. The dissociation constant, Kd,is given by: Kd=kd/ka=[Ag][Ab]/[Ag-Ab]. Kd has units of concentration,most typically M, mM, μM, nM, pM, etc. When comparing antibodyaffinities expressed as Kd, having greater affinity for NGF is indicatedby a lower value. The association constant, Ka, is given by:Ka=kaNd=[Ag−Ab]/[Ag][Ab]. Ka has units of inverse concentration, mosttypically M⁻¹, mM⁻¹, .μ.M⁻¹, nM⁻¹, pM⁻¹, etc. As used herein, the term“avidity” refers to the strength of the antigen-antibody bond afterformation of reversible complexes. Anti-NGF antibodies may becharacterized in terms of the Kd for their binding to a NGF protein, asbinding “with a dissociation constant (Kd) in the range of from about(lower Kd value) to about (upper Kd value).”

The terms “polypeptide”, “oligopeptide”, “peptide” and “protein” areused interchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, un-naturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon an antibody, the polypeptides can occur as single chains orassociated chains.

The term ‘conservative amino acid substitution” indicates any amino acidsubstitution for a given amino acid residue, where the substituteresidue is so chemically similar to that of the given residue that nosubstantial decrease in polypeptide function (e.g., enzymatic activity)results. Conservative amino acid substitutions are commonly known in theart and examples thereof are described, e.g., in U.S. Pat. Nos.6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferredembodiment, a conservative amino acid substitution will be anyone thatoccurs within one of the following six groups:

-   -   1. Small aliphatic, substantially non-polar residues: Ala, Gly,        Pro, Ser, and Thr;    -   2. Large aliphatic, non-polar residues: lie, Leu, and Val; Met;    -   3. Polar, negatively charged residues and their amides: Asp and        Glu;    -   4. Amides of polar, negatively charged residues: Asn and Gin;        His;    -   5. Polar, positively charged residues: Arg and Lys; His; and    -   6. Large aromatic residues: Trp and Tyr; Phe.

In a preferred embodiment, a conservative amino acid substitution willbe any one of the following, which are listed as Native Residue(Conservative Substitutions) pairs: Ala (Ser); Arg (Lys); Asn (Gin;His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn;Gin); lie(Leu; Val); Leu (lie; Val); Lys (Arg; Gin; Glu); Met (Leu; lie); Phe(Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe); andVal (lie; Leu).

The terms “nucleic acid”, “polynucleotide”, “nucleic acid molecule” andthe like may be used interchangeably herein and refer to a series ofnucleotide bases (also called “nucleotides”) in DNA and RNA. The nucleicacid may contain deoxyribonucleotides, ribonucleotides, and/or theiranalogs. The term “nucleic acid” includes, for example, single-strandedand double-stranded molecules. A nucleic acid can be, for example, agene or gene fragment, exons, introns, a DNA molecule (e.g., cDNA), anRNA molecule (e.g., mRNA), recombinant nucleic acids, plasmids, andother vectors, primers and probes. Both 5′ to 3′ (sense) and 3′ to 5′(antisense) polynucleotides are included. The nucleotides can bedeoxyribonucleotides, ribonucleotides, modified nucleotides or bases,and/or their analogs, or any substrate that can be incorporated into apolymer by DNA or RNA polymerase. A poly-nucleotide may comprisemodified nucleotides, such as methylated nucleotides and their analogs.If present, modification to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with alabeling component. Other types of modifications include, for example,“caps”, substitution of one or more of the naturally occurringnucleotides with an analog, internucleotide modifications such as, forexample, those with uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, cabamates, etc.) and with chargedlinkages (e.g., phosphorothioates, phosphorodithioates, etc.), thosecontaining pendant moieties, such as, for example, proteins (e.g.,nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.),those with intercalators (e.g., acridine, psoralen, etc.), thosecontaining chelators (e.g., metals, radioactive metals, boron, oxidativemetals, etc.), those containing alkylators, those with modified linkages(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified formsof the polynucleotide(s). Further, any of the hydroxyl groups ordinarilypresent in the sugars may be replaced, for example, by phosphonategroups, phosphate groups, protected by standard protecting groups, oractivated to prepare additional linkages to additional nucleotides, ormay be conjugated to solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupsmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-0-methyl-,2′-0-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses,pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs andabasic nucleoside analogs such as methyl riboside. One or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include, but are not limited to,embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR2 (”amidate“), P(O)R, P(O)OR', CO or CH2(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (-0-)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

As used herein, “vector” means a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a host cell. Examples of vectors include, butare not limited to, viral vectors, naked DNA or RNA expression vectors,plasmid, cosmid or phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells. Vectors, as described herein, have expression controlsequences meaning that a nucleic acid sequence that directstranscription of a nucleic acid. An expression control sequence can be apromoter, such as a constitutive or an inducible promoter, or anenhancer. The expression control sequence is ‘operably linked’ to thenucleic acid sequence to be transcribed. A nucleic acid is “operablylinked” when it is placed into a functional relationship with anothernucleic acid sequence. For example, DNA for a presequence or secretoryleader is operably linked to DNA for a polypeptide if it is expressed asa preprotein that participates in the secretion of the polypeptide; apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence; or a ribosome binding site isoperably linked to a coding sequence if it is positioned so as tofacilitate translation. Generally, “operably linked” means that the DNAsequences being linked are contiguous, and, in the case of a secretoryleader, contiguous and in reading phase. However, enhancers do not haveto be contiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accordance withconventional practice.

Just as a polypeptide may contain conservative amino acidsubstitution(s), a polynucleotide thereof may contain conservative codonsubstitution(s). A codon substitution is considered conservative if,when expressed, it produces a conservative amino acid substitution, asdescribed above. Degenerate codon substitution, which results in noamino acid substitution, is also useful in polynucleotides according tothe present invention. Thus, e.g., a polynucleotide encoding a selectedpolypeptide useful in an embodiment of the present invention may bemutated by degenerate codon substitution in order to approximate thecodon usage frequency exhibited by an expression host cell to betransformed therewith, or to otherwise improve the expression thereof.

A “variant” anti-NGF antibody, refers herein to a molecule which differsin amino acid sequence from a “parent” anti-NGF antibody amino acidsequence by virtue of addition, deletion, and/or substitution of one ormore amino acid residue(s) in the parent antibody sequence and retainsat least one desired activity of the parent anti-NGF-antibody. Thevariant anti-NGF may comprise conservative amino acid substitutions inthe hypervariable region of the antibody, as described herein. Desiredactivities can include the ability to bind the antigen specifically, theability to reduce, inhibit or neutralize NGF activity in an animal. Inone embodiment, the variant comprises one or more amino acidsubstitution(s) in one or more hypervariable and/or framework region(s)of the parent antibody. For example, the variant may comprise at leastone, e.g. from about one to about ten, and preferably from about two toabout five, substitutions in one or more hypervariable and/or frameworkregions of the parent antibody. Ordinarily, the variant will have anamino acid sequence having at least 50% amino acid sequence identitywith the parent antibody heavy or light chain variable domain sequences,more preferably at least 65%, more preferably at least 75%, morepreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, and most preferably at least 95% sequence identity.Identity or homology with respect to this sequence is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the parent antibody residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

None of N-terminal, C-terminal, or internal extensions, deletions, orinsertions into the antibody sequence shall be construed as affectingsequence identity or homology. The variant retains the ability to bindNGF and preferably has desired activities which are equal to or superiorto those of the parent antibody. For example, the variant may have astronger binding affinity, enhanced ability to reduce, inhibit orneutralize NGF activity in an animal, and/or enhanced ability to inhibitNGF binding to Trk A and p75.

Trk A, considered the high affinity NGF receptor is a member of theneurotrophic tyrosine kinase receptor (NTKR) family. This kinase is amembrane-bound receptor that, upon neurotrophin binding, phosphorylatesitself (autophosphorylation) and members of the MAPK pathway. Thepresence of this kinase leads to cell differentiation and may play arole in specifying sensory neuron subtypes. The p75 receptor isconsidered the low affinity NGF receptor.

A ‘variant” nucleic acid, refers herein to a molecule which differs insequence from a “parent” nucleic acid. Polynucleotide sequencedivergence may result from mutational changes such as deletions,substitutions, or additions of one or more nucleotides. Each of thesechanges may occur alone or in combination, one or more times in a givensequence.

The term “isolated” means that the material (e.g., antibody or nucleicacid) is separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials that would interfere with diagnostic or therapeutic uses forthe material, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. With respect to nucleic acid, an isolatednucleic acid may include one that is separated from the 5′ to 3′sequences with which it is normally associated in the chromosome. Inpreferred embodiments, the material will be purified to greater than 95%by weight of the material, and most preferably more than 99% by weight.Isolated material includes the material in situ within recombinant cellssince at least one component of the material's natural environment willnot be present. Ordinarily, however, isolated material will be preparedby at least one purification step.

As used herein, the terms “cell”, “cell line”, and “cell culture” may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell (e.g., bacterial cells,yeast cells, mammalian cells, and insect cells) whether located in vitroor in vivo. For example, host cells may be located in a transgenicanimal. Host cell can be used as a recipient for vectors and may includeany transformable organism that is capable of replicating a vectorand/or expressing a heterologous nucleic acid encoded by a vector.

The word “label” when used herein refers to a detectable compound orcomposition that is conjugated directly or indirectly to the antibody ornucleic acid. The label may itself be detectable by itself (e.g.,radioisotope labels or fluorescent labels) or, in the case of anenzymatic label, may catalyze chemical alteration of a substratecompound or composition that is detectable.

A “subject” or “patient” refers to an animal in need of treatment thatcan be affected by molecules of the invention. Animals that can betreated in accordance with the invention include vertebrates, withmammals such as canine being particularly preferred examples.

A “composition” is intended to mean a combination of active agent,whether chemical composition, biological composition or biotherapeutic(particularly antigen binding proteins as described herein) and anothercompound or composition which can be inert (e.g., a label), or active,such as an adjuvant.

As defined herein, “pharmaceutically acceptable carriers” suitable foruse in the invention are well known to those of skill in the art. Suchcarriers include, without limitation, water, saline, buffered saline,phosphate buffer, alcohol/aqueous solutions, emulsions or suspensions.Other conventionally employed diluents, adjuvants and excipients, may beadded in accordance with conventional techniques. Such carriers caninclude ethanol, polyols, and suitable mixtures thereof, vegetable oils,and injectable organic esters. Buffers and pH adjusting agents may alsobe employed. Buffers include, without limitation, salts prepared from anorganic acid or base. Representative buffers include, withoutlimitation, organic acid salts, such as salts of citric acid, e.g.,citrates, ascorbic acid, gluconic acid, histidine-Hel, carbonic acid,tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris,trimethanmine hydrochloride, or phosphate buffers. Parenteral carrierscan include sodium chloride solution, Ringer's dextrose, dextrose,trehalose, sucrose, and sodium chloride, lactated Ringer's or fixedoils. Intravenous carriers can include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose andthe like. Preservatives and other additives such as, for example,antimicrobials, antioxidants, chelating agents (e.g., EDTA), inert gasesand the like may also be provided in the pharmaceutical carriers. Thepresent invention is not limited by the selection of the carrier. Thepreparation of these pharmaceutically acceptable compositions, from theabove-described components, having appropriate pH isotonicity, stabilityand other conventional characteristics is within the skill of the art.See, e.g., texts such as Remington: The Science and Practice ofPharmacy, 20th ed, Lippincott Williams & Wilkins, pub!., 2000; and TheHandbook of Pharmaceutical Excipients, 4.sup.th edit., eds. R. C. Roweet al, APhA Publications, 2003.

A “therapeutically effective amount” (or “effective amount”) refers toan amount of an active ingredient, e.g., an agent according to theinvention, sufficient to effect beneficial or desired results whenadministered to a subject or patient. An effective amount can beadministered in one or more administrations, applications or dosages. Atherapeutically effective amount of a composition according to theinvention may be readily determined by one of ordinary skill in the art.In the context of this invention, a “therapeutically effective amount”is one that produces an objectively measured change in one or moreparameters associated NGF related condition sufficient to effectbeneficial or desired results including clinical results such asalleviation or reduction in pain sensation. An effective amount can beadministered in one or more administrations. For purposes of thisinvention, an effective amount of drug, compound, or pharmaceuticalcomposition is an amount sufficient to treat, ameliorate, reduce theintensity of and/or prevent pain, including post-surgical pain,rheumatoid arthritis pain, and/or osteoarthritis pain. In someembodiments, the “effective amount” may reduce pain at rest (restingpain) or mechanically- induced pain (including pain following movement),or both, and it may be administered before, during or after a painfulstimulus. As is understood in the clinical context, an effective amountof a drug, compound, or pharmaceutical composition may or may not beachieved in conjunction with another drug, compound, or pharmaceuticalcomposition. Thus, an “effective amount” may be considered in thecontext of administering one or more therapeutic agents, and a singleagent may be considered to be given in an effective amount if, inconjunction with one or more other agents, a desirable result may be oris achieved. Of course, the therapeutically effective amount will varydepending upon the particular subject and condition being treated, theweight and age of the subject, the severity of the condition, theparticular compound chosen, the dosing regimen to be followed, timing ofadministration, the manner of administration and the like, all of whichcan readily be determined by one of ordinary skill in the art.

As used herein, the term “therapeutic” encompasses the full spectrum oftreatments for a disease, condition or disorder. A “therapeutic” agentof the invention may act in a manner that is prophylactic or preventive,including those that incorporate procedures designed to target animalsthat can be identified as being at risk (pharmacogenetics); or in amanner that is ameliorative or curative in nature; or may act to slowthe rate or extent of the progression of at least one symptom of adisease or disorder being treated.

In a further aspect, the invention features veterinary compositions inwhich antibodies of the present invention are provided for therapeuticor prophylactic uses. The invention features a method for treating a dogsubject having a particular antigen, for example., one associated with adisease or condition. The method includes administering atherapeutically effective amount of a recombinant antibody specific forthe particular antigen, with the recombinant antibody described herein.

The amount of antibody useful to produce a therapeutic effect can bedetermined by standard techniques well known to those of ordinary skillin the art. The antibodies will generally be provided by standardtechnique within a pharmaceutically acceptable buffer, and may beadministered by any desired route. The route of administration of theantibody or antigen-binding moiety of the invention may be oral,parenteral, by inhalation or topical. In a preferred embodiment theroute of administration is parenteral. The term parenteral as usedherein includes intravenous, intramuscular, subcutaneous, rectal,vaginal or intraperitoneal administration.

“Pain” as used herein refers to pain of any etiology, including acuteand chronic pain, and any pain with an inflammatory component. Examplesof pain include including inflammatory pain, post-operative incisionpain, neuropathic pain, fracture pain, osteoporotic fracture pain,post-herpetic neuralgia, cancer pain, pain resulting from bums, painassociated with burn or wound, pain associated with trauma (includingtraumatic head injury), neuropathic pain, pain associated withmusculoskeletal disorders such as rheumatoid arthritis, osteoarthritis,ankylosing spondylitis, seronegative (non-rheumatoid) arthropathies,non-articular rheumatism and periarticular disorders, and painassociated with cancer (including “break-through pain” and painassociated with terminal cancer), peripheral neuropathy andpost-herpetic neuralgia.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, one or more ofthe following: improvement or alleviation of any aspect of pain,including acute, chronic, inflammatory, neuropathic, post-surgical pain,rheumatoid arthritis pain, or osteoarthritis pain. For purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, one or more of the following: including lessening severity,alleviation of one or more symptoms associated with pain including anyaspect of pain (such as shortening duration of pain, reduction of painsensitivity or sensation).

NGF Related Disorder, as described herein, refers to a disorderincluding cardiovascular diseases, atherosclerosis, obesity, type 2diabetes, metabolic syndrome, pain and inflammation. In some embodimentsof the present invention an NGF related disorder refers to pain, inparticular chronic pain, inflammatory pain, post-operative incisionpain, neuropathic pain, fracture pain, osteoporotic fracture pain,post-herpetic neuralgia, cancer pain, pain resulting from bums, painassociated with burn or wound, pain associated with trauma (includingtraumatic head injury), neuropathic pain, pain associated withmusculoskeletal disorders such as rheumatoid arthritis, osteoarthritis,ankylosing spondylitis, seronegative (non-rheumatoid) arthropathies,non-articular rheumatism and periarticular disorders, and painassociated with cancer (including “break-through pain” and painassociated with terminal cancer), peripheral neuropathy andpost-herpetic neuralgia. “Reducing incidence” of pain means any ofreducing severity (which can include reducing need for and/or amount of(e.g., exposure to) other drugs and/or therapies generally used for thisconditions, including, for example, opiates), duration, and/or frequency(including, for example, delaying or increasing time to post-surgicalpain in an individual). As is understood by those skilled in the art,individuals may vary in terms of their response to treatment, and, assuch, for example, a “method of reducing incidence of rheumatoidarthritis pain or osteoarthritis pain in an individual” reflectsadministering the anti-NGF antagonist antibody based on a reasonableexpectation that such administration may likely cause such a reductionin incidence in that particular individual.

“Ameliorating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means a lessening orimprovement of one or more symptoms of a pain as compared to notadministering an anti-NGF antagonist antibody. “Ameliorating” alsoincludes shortening or reduction in duration of a symptom.

“Palliating” a pain or one or more symptoms of a pain (such asrheumatoid arthritis pain or osteoarthritis pain) means lessening theextent of one or more undesirable clinical manifestations ofpost-surgical pain in an individual or population of individuals treatedwith an anti-NGF antagonist antibody in accordance with the invention.

As used therein, “delaying” the development of pain means to defer,hinder, slow, retard, stabilize, and/or postpone progression of pain,such as post-surgical pain, rheumatoid arthritis pain, or osteoarthritispain. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individuals being treated. As is evidentto one skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developpain. A method that “delays” development of the symptom is a method thatreduces probability of developing the symptom in a given time frameand/or reduces extent of the symptoms in a given time frame, whencompared to not using the method. Such comparisons are typically basedon clinical studies, using a statistically significant number ofsubjects.

“Post-surgical pain” (interchangeably termed “post-incisional” or“post-traumatic pain”) refers to pain arising or resulting from anexternal trauma such as a cut, puncture, incision, tear, or wound intotissue of an individual (including that that arises from all surgicalprocedures, whether invasive or non-invasive). As used herein,post-surgical pain does not include pain that occurs (arises ororiginates) without an external physical trauma. In some embodiments,post-surgical pain is internal or external (including peripheral) pain,and the wound, cut, trauma, tear or incision may occur accidentally (aswith a traumatic wound) or deliberately (as with a surgical incision).As used herein, “pain” includes nociception and the sensation of pain,and pain can be assessed objectively and subjectively, using pain scoresand other methods well-known in the art. Post-surgical pain, as usedherein, includes allodynia (i.e., increased response to a normallynon-noxious stimulus) and hyperalgesia (i.e., increased response to anormally noxious or unpleasant stimulus), which can in turn, be thermalor mechanical (tactile) in nature. In some embodiments, the pain ischaracterized by thermal sensitivity, mechanical sensitivity and/orresting pain. In some embodiments, the post-surgical pain comprisesmechanically-induced pain or resting pain. In other embodiments, thepost-surgical pain comprises resting pain. The pain can be primary orsecondary pain, as is well-known in the art.

Before the present methods are described, it is to be understood thatthis invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention will be limitedonly by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The invention disclosed herein concerns antibodies, which is usedinterchangeably with the term “antigen binding protein” as describedherein, that specifically bind to Nerve Growth Factor (NGF) and inparticular antibodies, whether it be canine antibodies produced byhybridoma or phage display technology or fully “caninized” monoclonalantibodies that specifically bind to canine NGF and thus prevent canineNGF from binding to canine TrkA and to a lesser extent canine p75receptors, thus serving as an antagonist in that the signaling pathwayis prevented from being activated by NGF.

NGF was the first neurotrophin to be identified, and its role in thedevelopment and survival of both peripheral and central neurons has beenwell characterized. NGF has been shown to be a critical survival andmaintenance factor in the development of peripheral sympathetic andembryonic sensory neurons and of basal forebrain cholinergic neurons(Smeyne et al. (1994) Nature 368:246-249; Crowley et al. (1994) Cell76:1001-1011). NGF upregulates expression of neuropeptides in sensoryneurons (Lindsay et al. (1989) Nature 337:362-364) and its activity ismediated through two different membrane-bound receptors, the TrkAreceptor and what is considered the low affinity p75 common neurotrophinreceptor.

NGF has been shown to be elevated in NGF related disorders in which anelevated amount of NGF is present in injured or diseased tissues. An NGFrelated disorder, can be defined as an increase in pain due to theelevation of NGF in an injured, diseased or damaged tissue. Pain, asused herein, is defined as described herein, refers to a disorderincluding chronic pain, inflammatory pain, post-operative incision pain,neuropathic pain, fracture pain, osteoporotic fracture pain,post-herpetic neuralgia, cancer pain, pain resulting from bums, painassociated with burn or wound, pain associated with trauma (includingtraumatic head injury), neuropathic pain, pain associated withmusculoskeletal disorders such as chronic pain, rheumatoid arthritis,osteoarthritis, ankylosing spondylitis, seronegative (non-rheumatoid)arthropathies, non-articular rheumatism and periarticular disorders, andpain associated with cancer (including “break-through pain” and painassociated with terminal cancer), peripheral neuropathy andpost-herpetic neuralgia.

In an embodiment of the present invention, an NGF disorder is defined asosteoarthritis in canines. Osteoarthritis (OA) is a slowly-progressivedegenerative joint disease characterized by a loss of joint cartilageand the subsequent exposure of subchondral bone in canines. Thiseventually results in a self-perpetuating insidious disordercharacterized by joint pain. New bone formation occurs in response tothe chronic inflammation, and local tissue damage in an attempt to limitboth movement and pain. Macroscopically, there is loss of jointcartilage, a narrowing of the joint space, sclerosis of subchondralbone, and the production of joint osteophytes (Veterinary Focus: Vol 17No 3; 2007)

In canines, the onset of primary OA depends on breed. The onset mean ageis 3.5 years in Rottweilers and 9.5 years in Poodles for examples, witha wide range of onset for other breeds as well as mixed breeds. Thedevelopmental orthopedic diseases and associated osteoarthritis are themost common articular diseases in dogs, they account for some 70% ofmedical visits for articular disease and related problems within theappendicular skeleton. Twenty two percent of cases were dogs aged oneyear or under. The incidence of OA is increased by trauma as well asobesity, aging and genetic abnormalities. In particular, age can be afactor in OA incidence wherein >50% of arthritis cases are observed indogs aged between 8-13 years. The musculoskeletal diseases are verycommon in geriatric patients, and nearly 20% of elderly dogs showorthopedic disorders. In Labrador Retrievers aged >8 years, OA inseveral joints (elbow, shoulder, hip, knee) is typical. Additionally thesize of the canine plays a role in OA onset as well. 45% of dogs witharthritis are large breed dogs. Among these, >50% are giant breed dogs,while only 28% are medium breed dogs and 27% are small breed dogs. Theneed for pharmaceutical intervention for the alleviation of OA pain incanines is very high.

As stated herein, elevated levels of NGF are indicative of a NGF relateddisorder, particularly in OA. Elevated levels of NGF have been reportedin transgenic arthritic mice along with an increase in the number ofmast cells (Aloe, et al., Int. J. Tissue Reactions-Exp. Clin. Aspects15:139-143 (1993)). PCT Publication No. WO 02/096458 discloses use ofanti NGF antibodies of certain properties in treating various NGFrelated disorders such as inflammatory condition (e.g., rheumatoidarthritis). It has been reported that a purified anti-NGF antibodyinjected into arthritic transgenic mice carrying the human tumornecrosis factor gene caused reduction in the number of mast cells, aswell as a decrease in histamine and substance P levels within thesynovium of arthritis mice (Aloe et al., Rheumatol. Int. 14: 249-252(1995)). It has been shown that exogenous administration of an NGFantibody reduced the enhanced level of TNFa, occurring in arthritic mice(Marmi et al., Rheumatol. Int. 18: 97-102 (1998)). Rodent anti-NGFantagonist antibodies have been reported. See, e.g., Hongo et al.,Hybridoma (2000) 19(3): 215- 227; Ruberti et al. (1993) Cell. Molec.Neurobiol. 13(5): 559-568. However, when rodent antibodies are usedtherapeutically in non-murine mammals, an anti-murine antibody responsedevelops in significant numbers of treated individuals. Thus, there is aserious need for anti-NGF antagonist antigen binding proteins, includinganti-NGF antagonist antibodies of the present invention for canine useparticularly for use in treating OA.

While the properties of antibodies make them very attractive therapeuticagents, there are a number of limitations. The vast majority ofmonoclonal antibodies (mAbs) are of rodent origin, as previously noted.When such antibodies are administered in a different species, patientscan mount their own antibody response to such xenogenic antibodies. Suchresponse may result in the eventual neutralization and elimination ofthe antibody. As described above mice are used extensively in theproduction of monoclonal antibodies. One problem in the use of usingantibodies produced by a particular species, generally initially in themouse, is that a non-murine subjects being treated with said antibodiesreact to the mouse antibodies as if they were a foreign substance thuscreating a new set of antibodies to the mouse antibodies. Mouseantibodies are “seen” by the non-murine, for example canine, immunesystem as foreign, and the subject then mounts an immune responseagainst the molecule. Those skilled in the field will recognize the needto be able to treat a subject with an antigen specific antibody, buthave that antibody species specific. Part of the reaction generated fromcross species antibody administration, for example a mouse monoclonalantibody being administered to a canine, can range from a mild form,like a rash, to a more extreme and life-threatening response, such asrenal failure. This immune response can also decrease the effectivenessof the treatment, or create a future reaction if the subject is given asubsequent treatment containing mouse antibodies. Accordingly, we setforth to overcome this disadvantage by “caninization” of an antibody. Inparticular, this process focuses on the framework regions of theimmunoglobulin variable domain, but could also include the complimentdeterminant regions (CDR's) of the variable domain. The enabling stepsand reduction to practice for this process are described in thisdisclosure.

The process of modifying a monoclonal antibody from an animal to renderit less immunogenic for therapeutic administration to species has beenaggressively pursued and has been described in a number of publications(e.g. Antibody Engineering: A practical Guide. Carl A. K. Borrebaeck ed.W. H. Freeman and Company, 1992). However, this process has not beenapplied for the development of therapeutic or diagnostics fornon-humans, particularly canines, until recently. In fact, very littlehas been published with regard to canine variable domains at all.Wasserman and Capra, Biochem. 6, 3160 (1977), determined the amino acidsequence of the variable regions of both a canine IgM and a canine IgAheavy chain. Wasserman and Capra, Immunochem. 15, 303 (1978), determinedthe amino acid sequence of the K light chain from a canine IgA. McCumberand Capra, Mol. Immunol. 16, 565 (1979), disclose the completeamino-acid sequence of a canine mu chain. Tang et al., Vet. ImmunologyImmunopathology 80, 259 (2001), discloses a single canine IgG-A y chaincDNA and four canine IgG-A y chain protein sequences. It describes PCRamplification of a canine spleen cDNA library with a degenerateoligonucleotide primer designed from the conserved regions of human,mouse, pig, and bovine IgGs. The paucity of information available oncanine antibodies has prevented their development as therapeutics forthe treatment canine disease.

These noted limitations have prompted the development of engineeringtechnologies known as “speciation” and is well known to those in the artin terms of “humanization” of therapeutic antibodies. Humanizedantibodies can be generated as chimeric antibodies or fragments thereofwhich contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human antibodies (i.e.“recipient antibody” or “target species antibody”) in which residuesfrom a complementarity determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (i.e. “donorantibody” or “originating species antibody”) such as mouse, having thedesired. properties such as specificity, affinity, and potency. In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. This humanizationstrategy is referred to as “CDR grafting” as reported for the making ofhumanized antibodies (Winter, U.S. Pat. No. 5,225,539). Back mutation ofselected target framework residues to the corresponding donor residuesmight be required to restore and or improved affinity. Structure-basedmethods may also be employed for humanization and affinity maturation,for example as described for humanization in U.S. patent applicationSer. No. 10/153,159 and related applications. Comparison of theessential framework residues required in humanization of severalantibodies, as well as computer modeling based on antibody crystalstructures revealed a set of framework residues termed as “Vernier zoneresidues” (Foote, J. Mol. Biol. 224:487-499 (1992)). In addition,several residues in the VH-VL interface zone have been suggested to beimportant in maintaining affinity for the antigen (Santos, Prog NucleicAcid Res Mol Biol. 60: 169-94 (1998); Kettleborough, et al., ProteinEngin., 4:773-783 (1991)). Similar strategies for “caninization” ofantibodies for use in dogs are described in U.S. Pat. No. 7,261,890.

Alternatively, humanized antibodies may contain the CDRs from anon-human sequence grafted into pools (e.g. libraries) of individualhuman framework regions. This newly engineered antibody is able to bindto the same antigen as the original antibody. The antibody constantregion is derived from a human antibody. The methodology for performingthis aspect is generally described as framework shuffling (Dall'Acqua,Methods, 36:43-60 (2005)). Furthermore, the humanized antibody maycontain sequences from two or more framework regions derived from atleast two human antibody germline sequences with high homology to thedonor species. Antibodies designed using this method are described ashybrid antibodies (Rother et al., U.S. Pat. No. 7,393,648) and may beapplicable to speciation outside of humanization, for example forcaninization.

The approaches described above utilize essentially entire frameworkregions from one or more antibody variable heavy chains or variablelight chains of the target species which are engineered to receive CDRsfrom the donor species. In some cases, back mutation of selectedresidues in the variable region is used to enhance presentation of theCDRs. Designing antibodies that minimize immunogenic reaction in asubject to non-native sequences in the antibody, while at the same timepreserving antigen binding regions of the antibody sufficiently tomaintain efficacy, has proven challenging.

Another challenge for developing therapeutic antibodies targetingproteins is that epitopes on the homologous protein in a differentspecies are frequently different, and the potential for cross-reactivitywith other proteins is also different. As a consequence, antibodies haveto be made, tested and developed for the specific target in theparticular species to be treated.

Antibodies target an antigen through its binding of a specific epitopeon an antigen by the interaction with the variable region of theantibody molecule. Furthermore, antibodies have the ability to mediate,inhibit (as in the case of the antagonistic anti-NGF antigen bindingprotein of the present invention) and/or initiate a variety ofbiological activities. There are a wide range of functions fortherapeutic antibodies, for example, antibodies can modulatereceptor-ligand interactions as agonists or antagonists. Antibodybinding can initiate intracellular signaling to stimulate cell growth,cytokine production, or apoptosis. Antibodies can deliver agents boundto the Fe region to specific sites. Antibodies also elicitantibody-mediated cytotoxicity (ADCC), complement-mediated cytotoxicity(CDC), and phagocytosis. There are also antibodies that have beenaltered where the ADCC, CDC, C1q binding and phagocytosis functions havebeen eliminated. In one embodiment of the present invention the antibodyof the present invention comprises alterations in the Fc region of theantibody that alters effector function of said antibody.

Caninization

As used herein, “caninized antibody” means an antibody having an aminoacid sequence corresponding to that of an antibody produced by a canineand/or has been made using any of the techniques known in the art ordisclosed herein. This definition of a caninized antibody includesantibodies comprising at least one canine heavy chain polypeptide or atleast one canine light chain polypeptide. “Speciation”, per se, ofantibodies, and in particular the humanization of antibodies is a fieldof study well known to one skilled in the art. It has been unknown untilrecently whether the speciation of antibodies beyond humanization wouldyield a therapeutic antibody that could be efficacious in any otherspecies. The present invention exemplifies the caninization of ananti-NGF antibody for therapeutic use in dogs.

Chimeric antibodies comprise sequences from at least two differentspecies. As one example, recombinant cloning techniques may be used toinclude variable regions, which contain the antigen-binding sites, froma non-canine antibody (i.e., an antibody prepared in a non-caninespecies immunized with the antigen) and constant regions derived from acanine immunoglobulin.

Caninized antibodies are a type of chimeric antibody wherein variableregion residues responsible for antigen binding (i.e., residues of acomplementarity determining region, abbreviated complementaritydetermining region, or any other residues that participate in antigenbinding) are derived from a non-canine species, while the remainingvariable region residues (i.e., residues of the framework regions) andconstant regions are derived, at least in part, from canine antibodysequences. A subset of framework region residues and constant regionresidues of a caninized antibody may be derived from non-canine sources.Variable regions of a caninized antibody are also described as caninized(i.e., a caninized light or heavy chain variable region). The non-caninespecies is typically that used for immunization with antigen, such asmouse, rat, rabbit, non-human primate, or other non-canine mammalianspecies.

Complementarity determining regions (CDRs) are residues of antibodyvariable regions that participate in antigen binding. Several numberingsystems for identifying CDRs are in common use. The Kabat definition isbased on sequence variability, and the Clothia definition is based onthe location of the structural loop regions. The AbM definition is acompromise between the Kabat and Clothia approaches. A caninizedantibody of the invention may be constructed to comprise one or moreCDRs. Still further, CDRs may be used separately or in combination insynthetic molecules such as SMIPs and small antibody mimetics.

Framework residues are those residues of antibody variable regions otherthan hypervariable or CDR residues. Framework residues may be derivedfrom a naturally occurring canine antibody, such as a canine frameworkthat is substantially similar to a framework region of the antibody ofthe invention. Artificial framework sequences that represent a consensusamong individual sequences may also be used. When selecting a frameworkregion for caninization, sequences that are widely represented incanines may be preferred over less populous sequences. Additionalmutations of the canine framework acceptor sequences may be made torestore murine residues believed to be involved in antigen contactsand/or residues involved in the structural integrity of theantigen-binding site, or to improve antibody expression.

Grafting of CDRs is performed by replacing one or more CDRs of anacceptor antibody (e.g., a caninized antibody or other antibodycomprising desired framework residues) with CDRs of a donor antibody(e.g., a non-canine antibody). Acceptor antibodies may be selected basedon similarity of framework residues between a candidate acceptorantibody and a donor antibody. For example, canine framework regions areidentified as having substantial sequence homology to each frameworkregion of the relevant non-canine antibody, and CDRs of the non-canineantibody are grafted onto the composite of the different canineframework regions.

Analysis of the three-dimensional structures of antibody-antigencomplexes, combined with analysis of the available amino acid sequencedata may be used to model sequence variability based on structuraldissimilarity of amino acid residues that occur at each position withinthe CDR. CDRs of the present invention can also be utilized in smallantibody mimetics, which comprise two CDR regions and a framework region(Qui et al. Nature Biotechnology Vol 25;921-929; August 2007).

Acceptor frameworks for grafting of CDRs or abbreviated CDRs may befurther modified to introduce desired residues. For example, acceptorframeworks may comprise a heavy chain variable region of a canineconsensus sequence, optionally with non-canine donor residues at one ormore of positions. Following grafting, additional changes may be made inthe donor and/or acceptor sequences to optimize antibody binding andfunctionality. See e.g., International Publication No. WO 91/09967.

The present invention further provides cells and cell lines expressingantibodies of the invention. Representative host cells includebacterial, yeast, mammalian and human cells, such as CHO cells, HEK-293cells, HeLa cells, CV-1 cells, and COS cells. Methods for generating astable cell line following transformation of a heterologous constructinto a host cell are known in the art. Representative non-mammalian hostcells include insect cells (Potter et al. (1993) Int. Rev. Immunol.10(2-3):103-112). Antibodies may also be produced in transgenic animals(Houdebine (2002) Curr. Opin. Biotechnol. 13(6):625-629) and transgenicplants (Schillberg et al. (2003) Cell Mol. Life Sci. 60(3):433-45).

As discussed above, monoclonal, chimeric and caninized antibodies, whichhave been modified by, e.g., deleting, adding, or substituting otherportions of the antibody, e.g., the constant region, are also within thescope of the invention. For example, an antibody can be modified asfollows: (i) by deleting the constant region; (ii) by replacing theconstant region with another constant region, e.g., a constant regionmeant to increase half-life, stability or affinity of the antibody, or aconstant region from another species or antibody class; or (iii) bymodifying one or more amino acids in the constant region to alter, forexample, the number of glycosylation sites, effector cell function, Fcreceptor (FcR) binding, complement fixation, among others. In oneembodiment of the present invention the antibody of the inventioncomprises an altered Fc region that alters effector function of theantibody.

Methods for altering an antibody constant region are known in the art.Antibodies with altered function, e.g. altered affinity for an effectorligand, such as FcR on a cell, or the C1 component of complement can beproduced by replacing at least one amino acid residue in the constantportion of the antibody with a different residue (see e.g., EP 388,151A1, U.S. Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260, the contents ofall of which are hereby incorporated by reference).

For example, it is possible to alter the affinity of an Fc region of anantibody for an FcR (e.g., Fc.gamma.R1), or for C1q binding by replacingthe specified residue(s) with a residue(s) having an appropriatefunctionality on its side chain, or by introducing a charged functionalgroup, such as glutamate or aspartate, or perhaps an aromatic non-polarresidue such as phenylalanine, tyrosine, tryptophan or alanine (seee.g., U.S. Pat. No. 5,624,821). The antibody or binding fragment thereofmay be conjugated with a cytotoxin, a therapeutic agent, or aradioactive metal ion. In one embodiment, the protein that is conjugatedis an antibody or fragment thereof. A cytotoxin or cytotoxic agentincludes any agent that is detrimental to cells. Non-limiting examplesinclude, calicheamicin, taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, and analogs, or homologs thereof. Therapeutic agents include,but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP), cisplatin),anthracyclines (e.g., daunorubicin and doxorubicin), antibiotics (e.g.,dactinomycin, bleomycin, mithramycin, and anthramycin), and anti-mitoticagents (e.g., vincristine and vinblastine). Techniques for conjugatingsuch moieties to proteins are well known in the art.

Compositions, Derived Compositions, and Methods of Making theCompositions

This invention encompasses compositions, including pharmaceuticalcompositions, comprising antibodies, polypeptides and polynucleotidescomprising sequences encoding antibodies or polypeptides of theinvention.

As used herein, compositions comprise one or more antibodies, antigenbinding proteins or polypeptides (which may or may not be an antibody)that bind to canine NGF, and/or one or more polynucleotides comprisingsequences encoding one or more antibodies or polypeptides that bind toNGF. These compositions may further comprise suitable excipients, suchas pharmaceutically acceptable excipients including buffers, which arewell known in the art. The invention also encompasses isolated antibody,polypeptide and polynucleotide embodiments. The invention alsoencompasses substantially pure antibody, polypeptide and polynucleotideembodiments.

In some embodiments, the present invention provides for novel caninizedmonoclonal antibodies that specifically bind to canine NGF. In oneembodiment, a monoclonal antibody of the invention binds to NGF andprevents its binding to, and activation of, its receptors Trk A and to alesser extent p75, thus preventing the signaling cascade as describedherein. The monoclonal antibodies of the present invention areidentified herein as “SM57”, “SM58”, “SM66”, “CanE3M65-12” and“CANSSM-QC23-VL/CANSSM57-VH” (“SSMQC23HCLC”)

In one or more embodiments, the present invention provides an isolatedcaninized antigen binding protein wherein the variable light chaincomprises SEQ ID NO. 16 (CAN-E3M-VL) and the variable heavy chaincomprises SEQ ID NO. 17 (CAN-N2G9-VH).

In one or more embodiments, the present invention provides an isolatedcaninized antigen binding protein comprising a variable light chaincomprising SEQ ID NO. 26 (CAN-SSME3M-VL) and the variable heavy chaincomprising SEQ ID NO. 27 (CAN-SSM57-VH).

In one or more embodiments, the present invention provides an isolatedcaninized antigen binding protein comprising a variable light chaincomprising SEQ ID NO. 30 (CAN-QC23-VL) and a variable heavy chaincomprising SEQ ID NO. 27 (CAN-SSM57-VH).

A further embodiment of the invention provides the nucleic acids thatencode the various antigen binding proteins as previously described.

The present invention provides for recombinant monoclonal antibodies andpeptides and their uses in clinical administrations and scientificprocedures, including diagnostic procedures. With the advent of methodsof molecular biology and recombinant technology, it is possible toproduce antibody and antibody-like molecules by recombinant means andthereby generate gene sequences that code for specific amino acidsequences found in the polypeptide structure of the antibodies. Suchantibodies can be produced by either cloning the gene sequences encodingthe polypeptide chains of said antibodies or by direct synthesis of saidpolypeptide chains, with assembly of the synthesized chains to formactive tetrameric (H2L2) structures with affinity for specific epitopesand antigenic determinants. This has permitted the ready production ofantibodies having sequences characteristic of neutralizing antibodiesfrom different species and sources.

Regardless of the source of the antibodies, or how they arerecombinantly constructed, or how they are synthesized, in vitro or invivo, using transgenic animals, large cell cultures of laboratory orcommercial size, using transgenic plants, or by direct chemicalsynthesis employing no living organisms at any stage of the process, allantibodies have a similar overall 3 dimensional structure. Thisstructure is often given as H2L2 and refers to the fact that antibodiescommonly comprise two light (L) amino acid chains and 2 heavy (H) aminoacid chains. Both chains have regions capable of interacting with astructurally complementary antigenic target. The regions interactingwith the target are referred to as “variable” or ‘V” regions and arecharacterized by differences in amino acid sequence from antibodies ofdifferent antigenic specificity. The variable regions of either H or Lchains contain the amino acid sequences capable of specifically bindingto antigenic targets.

As used herein, the term “antigen binding region” refers to that portionof an antibody molecule which contains the amino acid residues thatinteract with an antigen and confer on the antibody its specificity andaffinity for the antigen. The antibody binding region includes the“framework” amino acid residues necessary to maintain the properconformation of the antigen-binding residues. Within the variableregions of the H or L chains that provide for the antigen bindingregions are smaller sequences dubbed “hypervariable” because of theirextreme variability between antibodies of differing specificity. Suchhypervariable regions are also referred to as “complementaritydetermining regions” or “CDR” regions. These CDR regions account for thebasic specificity of the antibody for a particular antigenic determinantstructure.

The CDRs represent non-contiguous stretches of amino acids within thevariable regions but, regardless of species, the positional locations ofthese critical amino acid sequences within the variable heavy and lightchain regions have been found to have similar locations within the aminoacid sequences of the variable chains. The variable heavy and lightchains of all antibodies each have three CDR regions, eachnon-contiguous with the others. In all mammalian species, antibodypeptides contain constant (i.e., highly conserved) and variable regions,and, within the latter, there are the CDRs and the so-called “frameworkregions” made up of amino acid sequences within the variable region ofthe heavy or light chain but outside the CDRs.

The present invention further provides a vector including at least oneof the nucleic acids described above. Because the genetic code isdegenerate, more than one codon can be used to encode a particular aminoacid. Using the genetic code, one or more different nucleotide sequencescan be identified, each of which would be capable of encoding the aminoacid. The probability that a particular oligonucleotide will, in fact,constitute the actual encoding sequence can be estimated by consideringabnormal base pairing relationships and the frequency with which aparticular codon is actually used (to encode a particular amino acid) ineukaryotic or prokaryotic cells expressing an anti-NGF antibody orportion. Such “codon usage rules” are disclosed by Lathe, et al., 183 J.Molec. Biol. 1-12 (1985). Using the “codon usage rules” of Lathe, asingle nucleotide sequence, or a set of nucleotide sequences thatcontains a theoretical “most probable” nucleotide sequence capable ofencoding anti-NGF sequences can be identified. It is also intended thatthe antibody coding regions for use in the present invention could alsobe provided by altering existing antibody genes using standard molecularbiological techniques that result in variants (agonists) of theantibodies and peptides described herein. Such variants include, but arenot limited to deletions, additions and substitutions in the amino acidsequence of the anti-NGF antibodies or peptides.

For example, one class of substitutions is conservative amino acidsubstitutions. Such substitutions are those that substitute a givenamino acid in an anti-NGF antibody peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and lie; interchange of the hydroxyl residues Ser and Thr, exchangeof the acidic residues Asp and Glu, substitution between the amideresidues Asn and Gin, exchange of the basic residues Lys and Arg,replacements among the aromatic residues Phe, Tyr, and the like.Guidance concerning which amino acid changes are likely to bephenotypically silent is found in Bowie et al., 247 Science 1306-10(1990).

Variant or agonist anti-NGF antibodies or peptides may be fullyfunctional or may lack function in one or more activities. Fullyfunctional variants typically contain only conservative variations orvariations in non-critical residues or in non-critical regions.Functional variants can also contain substitution of similar amino acidsthat result in no change or an insignificant change in function.Alternatively, such substitutions may positively or negatively affectfunction to some degree. Non-functional variants typically contain oneor more non-conservative amino acid substitutions, deletions,insertions, inversions, or truncation or a substitution, insertion,inversion, or deletion in a critical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis. Cunningham et al., 244 Science 1081-85 (1989). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as epitope binding or in vitro ADCC activity. Sites thatare critical for ligand-receptor binding can also be determined bystructural analysis such as crystallography, nuclear magnetic resonance,or photoaffinity labeling. Smith et al., 224 J. Mol. Biol. 899-904(1992); de Vos et al., 255 Science 306-12 (1992).

Moreover, polypeptides often contain amino acids other than the twenty“naturally occurring” amino acids. Further, many amino acids, includingthe terminal amino acids, may be modified by natural processes, such asprocessing and other post-translational modifications, or by chemicalmodification techniques well known in the art. Known modificationsinclude, but are not limited to, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent crosslinks, formation of cystine, formation of pyroglutamate,formylation, gamma carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill in the art and have been described ingreat detail in the scientific literature. Several particularly commonmodifications, glycosylation, lipid attachment, sulfation,gamma-carboxylation of glutamic acid residues, hydroxylation and ADPribosylation, for instance, are described in most basic texts, such asProteins-Structure and Molecular Properties (2nd ed., T. E. Creighton,W. H. Freeman & Co., N.Y., 1993). Many detailed reviews are available onthis subject, such as by Wold, Posttranslational Covalent Modificationof proteins, 1-12 (Johnson, ed., Academic Press, N.Y., 1983); Seifter etal. 182 Meth. Enzymol. 626-46 (1990); and Rattan et al. 663 Ann. NYAcad. Sci. 48-62 (1992).

Accordingly, the antibodies and peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code. Similarly, the additionsand substitutions in the amino acid sequence as well as variations, andmodifications just described may be equally applicable to the amino acidsequence of the NGF antigen and/or epitope or peptides thereof, and arethus encompassed by the present invention. As mentioned above, the genesencoding a monoclonal antibody according to the present invention isspecifically effective in the recognition of NGF.

Antibody Derivatives

Included within the scope of this invention are antibody derivatives. A“derivative” of an antibody contains additional chemical moieties notnormally a part of the protein. Covalent modifications of the proteinare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the antibody with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues. For example,derivatization with bifunctional agents, well-known in the art, isuseful for cross-linking the antibody or fragment to a water-insolublesupport matrix or to other macromolecular carriers.

Derivatives also include radioactively labeled monoclonal antibodiesthat are labeled. For example, with radioactive iodine (251,1311),carbon (4C), sulfur (35S), indium, tritium (H³) or the like; conjugatesof monoclonal antibodies with biotin or avidin, with enzymes, such ashorseradish peroxidase, alkaline phosphatase, beta-D-galactosidase,glucose oxidase, glucoamylase, carboxylic acid anhydrase, acetylcholineesterase, lysozyme, malate dehydrogenase or glucose 6-phosphatedehydrogenase; and also conjugates of monoclonal antibodies withbioluminescent agents (such as luciferase), chemoluminescent agents(such as acridine esters) or fluorescent agents (such asphycobiliproteins).

Another derivative bifunctional antibody of the present invention is abispecific antibody, generated by combining parts of two separateantibodies that recognize two different antigenic groups. This may beachieved by crosslinking or recombinant techniques. Additionally,moieties may be added to the antibody or a portion thereof to increasehalf-life in vivo (e.g., by lengthening the time to clearance from theblood stream. Such techniques include, for example, adding PEG moieties(also termed pegilation), and are well-known in the art. See U.S.Patent. Appl. Pub. No. 20030031671.

Recombinant Expression of Antibodies

In some embodiments, the nucleic acids encoding a subject monoclonalantibody are introduced directly into a host cell, and the cell isincubated under conditions sufficient to induce expression of theencoded antibody. After the subject nucleic acids have been introducedinto a cell, the cell is typically incubated, normally at 37° C.,sometimes under selection, for a period of about 1-24 hours in order toallow for the expression of the antibody. In one embodiment, theantibody is secreted into the supernatant of the media in which the cellis growing. Traditionally, monoclonal antibodies have been produced asnative molecules in murine hybridoma lines. In addition to thattechnology, the present invention provides for recombinant DNAexpression of monoclonal antibodies. This allows the production ofcaninized antibodies, as well as a spectrum of antibody derivatives andfusion proteins in a host species of choice.

A nucleic acid sequence encoding at least one anti-NGF antibody, portionor polypeptide of the present invention may be recombined with vectorDNA in accordance with conventional techniques, including blunt-ended orstaggered-ended termini for ligation, restriction enzyme digestion toprovide appropriate termini, filling in of cohesive ends as appropriate,alkaline phosphatase treatment to avoid undesirable joining, andligation with appropriate ligases. Techniques for such manipulations aredisclosed, e.g., by Maniatis et al., MOLECULAR CLONING, LAB. MANUAL,(Cold Spring Harbor Lab. Press, NY, 1982 and 1989), and Ausubel et al.1993 supra, may be used to construct nucleic acid sequences which encodea monoclonal antibody molecule or antigen binding region thereof.

A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene expression as anti-NGFpeptides or antibody portions in recoverable amounts. The precise natureof the regulatory regions needed for gene expression may vary fromorganism to organism, as is well known in the analogous art. See, e.g.,Sambrook et al., 2001 supra; Ausubel et al., 1993 supra.

The present invention accordingly encompasses the expression of ananti-NGF antibody or peptide, in either prokaryotic or eukaryotic cells.Suitable hosts include bacterial or eukaryotic hosts including bacteria,yeast, insects, fungi, bird and mammalian cells either in vivo, or insitu, or host cells of mammalian, insect, bird or yeast origin. Themammalian cell or tissue may be of human, primate, hamster, rabbit,rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any othermammalian cell may be used.

In one embodiment, the nucleotide sequence of the invention will beincorporated into a plasmid or viral vector capable of autonomousreplication in the recipient host. Any of a wide variety of vectors maybe employed for this purpose. See, e.g., Ausubel et al., 1993 supra.Factors of importance in selecting a particular plasmid or viral vectorinclude: the ease with which recipient cells that contain the vector maybe recognized and selected from those recipient cells which do notcontain the vector; the number of copies of the vector which are desiredin a particular host; and whether it is desirable to be able to“shuttle” the vector between host cells of different species.

Example prokaryotic vectors known in the art include plasmids such asthose capable of replication in E. coli (such as, for example, pBR322,CoIE1, pSC101, pACYC 184, .pi.vX). Such plasmids are, for example,disclosed by Maniatis et aI., 1989 supra; Ausubel et al, 1993 supra.Bacillus plasmids include pC194, pC221 , pT127, etc. Such plasmids aredisclosed by Gryczan, in THE MOLEC. BIO. OF THE BACILLI 307-329(Academic Press, NY, 1982). Suitable Streptomyces plasmids includep1J101 (Kendall et al., 169 J. Bacteriol. 4177-83 (1987), andStreptomyces bacteriophages such as phLC31 (Chater et al., in SIXTHINT'L SYMPOSIUM ON ACTINOMYCETALES BIO. 45-54 (Akademiai Kaido,Budapest, Hungary 1986). Pseudomonas plasmids are reviewed in John etaI., 8 Rev. Infect. Dis. 693-704 (1986); lzaki, 33 Jpn. J. Bacteriol.729-42 (1978); and Ausubel et aI., 1993 supra.

Alternatively, gene expression elements useful for the expression ofcDNA encoding anti-NGF antibodies or peptides include, but are notlimited to (a) viral transcription promoters and their enhancerelements, such as the SV40 early promoter (Okayama et aI., 3 Mol. Cell.Biol. 280 (1983), Rous sarcoma virus LTR (Gorman et aI., 79 Proc. Natl.Acad. Sci., USA 6777 (1982), and Moloney murine leukemia virus LTR(Grosschedl et aI., 41 Cell 885 (1985); (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region(Okayarea et aI., 1983), and (c) polyadenylation sites such as in SV40(Okayama et aI., 1983).

Immunoglobulin cDNA genes can be expressed as described by Weidle etaI., 51 Gene 21 (1987), using as expression elements the SV40 earlypromoter and its enhancer, the mouse immunoglobulin H chain promoterenhancers, SV40 late region mRNA splicing, rabbit S-globin interveningsequence, immunoglobulin and rabbit S- globin polyadenylation sites, andSV40 polyadenylation elements. For immunoglobulin genes comprised ofpart cDNA, part genomic DNA (Whittle et aI., 1 Protein Engin. 499(1987>, the transcriptional promoter can be human cytomegalovirus, thepromoter enhancers can be cytomegalovirus and mouse/humanimmunoglobulin, and mRNA splicing and polyadenylation regions can be thenative chromosomal immunoglobulin sequences.

In one embodiment, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enhancers are either or both the mouse immunoglobulin heavychain enhancer and the viral LTR enhancer, the splice region contains anintron of greater than 31 bp, and the polyadenylation and transcriptiontermination regions are derived from the native chromosomal sequencecorresponding to the immunoglobulin chain being synthesized. In otherembodiments, cDNA sequences encoding other proteins are combined withthe above-recited expression elements to achieve expression of theproteins in mammalian cells.

Each fused gene can be assembled in, or inserted into, an expressionvector. Recipient cells capable of expressing the chimericimmunoglobulin chain gene product are then transfected singly with ananti-NGF peptide or chimeric H or chimeric L chain-encoding gene, or areco-transfected with a chimeric H and a chimeric L chain gene. Thetransfected recipient cells are cultured under conditions that permitexpression of the incorporated genes and the expressed immunoglobulinchains or intact antibodies or fragments are recovered from the culture.

In one embodiment, the fused genes encoding the anti-NGF peptide orchimeric H and L chains, or portions thereof are assembled in separateexpression vectors that are then used to cotransfect a recipient cell.Alternatively the fused genes encoding the chimeric H and L chains canbe assembled on the same expression vector. For transfection of theexpression vectors and production of the chimeric antibody, therecipient cell line may be a myeloma cell. Myeloma cells can synthesize,assemble and secrete immunoglobulins encoded by transfectedimmunoglobulin genes and possess the mechanism for glycosylation of theimmunoglobulin. Myeloma cells can be grown in culture or in theperitoneal cavity of a mouse, where secreted immunoglobulin can beobtained from ascites fluid. Other suitable recipient cells includelymphoid cells such as B lymphocytes of human or nonhuman origin,hybridoma cells of human or non-human origin, or interspeciesheterohybridoma cells.

The expression vector carrying a chimeric, caninized antibody constructor anti-NGF polypeptide of the present invention can be introduced intoan appropriate host cell by any of a variety of suitable means,including such biochemical means as transformation, transfection,conjugation, protoplast fusion, calcium phosphate- precipitation, andapplication with polycations such as diethylaminoethyl (DEAE) dextran,and such mechanical means as electroporation, direct microinjection, andmicroprojectile bombardment. Johnston et at, 240 Science 1538 (1988).

Yeast can provide substantial advantages over bacteria for theproduction of immunoglobulin H and L chains. Yeasts carry outpost-translational peptide modifications including glycosylation. Anumber of recombinant DNA strategies now exist which utilize strongpromoter sequences and high copy number plasmids which can be used forproduction of the desired proteins in yeast. Yeast recognizes leadersequences of cloned mammalian gene products and secretes peptidesbearing leader sequences (i.e., pre-peptides). Hitzman et al., 11thInt'l Conference on Yeast, Genetics & Molec. Biol. (Montpelier, France,1982).

Yeast gene expression systems can be routinely evaluated for the levelsof production, secretion and the stability of anti-NGF peptides,antibody and assembled murine and chimeric, heterochimeric, caninized,antibodies, fragments and regions thereof. Any of a series of yeast geneexpression systems incorporating promoter and termination elements fromthe actively expressed genes coding for glycolytic enzymes produced inlarge quantities when yeasts are grown in media rich in glucose can beutilized. Known glycolytic genes can also provide very efficienttranscription control signals. For example, the promoter and terminatorsignals of the phosphoglycerate kinase (PGK) gene can be utilized. Anumber of approaches can be taken for evaluating optimal expressionplasmids for the expression of cloned immunoglobulin cDNAs in yeast. SeeVol. II DNA Cloning, 45-66, (Glover, ed.,) IRL Press, Oxford, UK 1985).

Bacterial strains can also be utilized as hosts for the production ofantibody molecules or peptides described by this invention. Plasmidvectors containing replicon and control sequences which are derived fromspecies compatible with a host cell are used in connection with thesebacterial hosts. The vector carries a replication site, as well asspecific genes which are capable of providing phenotypic selection intransformed cells. A number of approaches can be taken for evaluatingthe expression plasmids for the production of murine, chimeric,heterochimeric, caninized antibodies, fragments and regions or antibodychains encoded by the cloned immunoglobulin cDNAs in bacteria (seeGlover, 1985 supra; Ausubel, 1993 supra; Sambrook, 2001 supra; Colliganet al., eds. Current Protocols in Immunology, John Wiley & Sons, NY,N.Y. (1994-2001); Colligan et al., eds. Current Protocols in ProteinScience, John Wiley & Sons, NY, N.Y. (1997-2001).

Host mammalian cells may be grown in vitro or in vivo. Mammalian cellsprovide posttranslational modifications to immunoglobulin proteinmolecules including leader peptide removal, folding and assembly of HandL chains, glycosylation of the antibody molecules, and secretion offunctional antibody protein. Mammalian cells which can be useful ashosts for the production of antibody proteins, in addition to the cellsof lymphoid origin described above, include cells of fibroblast origin,such as Vero (ATCC CRL 81) or CHO-K1 (ATCC CRL 61) cells. Many vectorsystems are available for the expression of cloned anti-NGF peptidesHand L chain genes in mammalian cells (see Glover, 1985 supra).Different approaches can be followed to obtain complete H2L2 antibodies.It is possible to co-express Hand L chains in the same cells to achieveintracellular association and linkage of Hand L chains into completetetrameric H2L2 antibodies and/or anti-NGF peptides. The co-expressioncan occur by using either the same or different plasmids in the samehost. Genes for both Hand L chains and/or anti- NGF peptides can beplaced into the same plasmid, which is then transfected into cells,thereby selecting directly for cells that express both chains.Alternatively, cells can be transfected first with a plasmid encodingone chain, for example the L chain, followed by transfection of theresulting cell line with an H chain plasmid containing a secondselectable marker. cell lines producing anti-NGF peptides and/or H2L2molecules via either route could be transfected with plasmids encodingadditional copies of peptides, H, L, or H plus L chains in conjunctionwith additional selectable markers to generate cell lines with enhancedproperties, such as higher production of assembled H2L2 antibodymolecules or enhanced stability of the transfected cell lines.

For long-term, high-yield production of recombinant antibodies, stableexpression may be used. For example, cell lines, which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with immunoglobulin expression cassettes and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into a chromosome and grow to form foci which inturn can be cloned and expanded into cell lines. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds/components that interact directly or indirectly with theantibody molecule.

Once an antibody of the invention has been produced, it may be purifiedby any method known in the art for purification of an immunoglobulinmolecule, for example, by chromatography (e.g., ion exchange, affinity,particularly affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins. Inmany embodiments, antibodies are secreted from the cell into culturemedium and harvested from the culture medium.

Pharmaceutical and Veterinary Applications

The anti-NGF antibodies or peptides of the present invention can be usedfor example in the treatment of NGF related disorders in dogs. Morespecifically, the invention further provides for a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier or diluentand, as active ingredient, an antibody or peptide according to theinvention. The antibody can be a chimeric, heterochimeric, caninized, orantibody according to the present invention. Intact immunoglobulins ortheir binding fragments, such as Fab, are also envisioned. The antibodyand pharmaceutical compositions thereof of this invention are useful forparenteral administration, e.g., subcutaneously, intramuscularly orintravenously.

Anti-NGF antibodies and/or peptides of the present invention can beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. They can be administered alone, but aregenerally administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice. Administration of the antibodies disclosed herein may becarried out by any suitable means, including parenteral injection (suchas intraperitoneal, subcutaneous, or intramuscular injection), orally,or by topical administration of the antibodies (typically carried in apharmaceutical formulation) to an airway surface. Topical administrationto an airway surface can be carried out by intranasal administration(e.g., by use of dropper, swab, or inhaler). Topical administration ofthe antibodies to an airway surface can also be carried out byinhalation administration, such as by creating respirable particles of apharmaceutical formulation (including both solid and liquid particles)containing the antibodies as an aerosol suspension, and then causing thesubject to inhale the respirable particles. Methods and apparatus foradministering respirable particles of pharmaceutical formulations arewell known, and any conventional technique can be employed.

In some desired embodiments, the antibodies are administered byparenteral injection. For parenteral administration, anti-NGF antibodiesor peptides can be formulated as a solution, suspension, emulsion orlyophilized powder in association with a pharmaceutically acceptableparenteral vehicle. For example the vehicle may be a solution of theantibody or a cocktail thereof dissolved in an acceptable carrier, suchas an aqueous carrier such vehicles are water, saline, Ringer'ssolution, dextrose solution, trehalose or sucrose solution, or 5% serumalbumin, 0.4% saline, 0.3% glycine and the like. Liposomes andnonaqueous vehicles such as fixed oils can also be used. These solutionsare sterile and generally free of particulate matter. These compositionsmay be sterilized by conventional, well known sterilization techniques.The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, toxicity adjustment agents and thelike, for example sodium acetate, sodium chloride, potassium chloride,calcium chloride, sodium lactate, etc. The concentration of antibody inthese formulations can vary widely, for example from less than about0.5%, usually at or at least about 1% to as much as 15% or 20% by weightand will be selected primarily based on fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.The vehicle or lyophilized powder can contain additives that maintainisotonicity (e.g., sodium chloride, mannitol) and chemical stability(e.g., buffers and preservatives). The formulation is sterilized bycommonly used techniques. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in, for example, REMINGTON'SPHARMA. SCI. (15th ed., Mack Pub. Co., Easton, Pa., 1980).

The antibodies of this invention can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immune globulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate. Thecompositions containing the present antibodies or a cocktail thereof canbe administered for prevention of recurrence and/or therapeutictreatments for existing disease. Suitable pharmaceutical carriers aredescribed in the most recent edition of REMINGTON'S PHARMACEUTICALSCIENCES, a standard reference text in this field of art. In therapeuticapplication, compositions are administered to a subject alreadysuffering from a disease, in an amount sufficient to cure or at leastpartially arrest or alleviate the disease and its complications. Anamount adequate to accomplish this is defined as a “therapeuticallyeffective dose” or a “therapeutically effective amount”. Amountseffective for this use will depend upon the severity of the disease andthe general state of the subject's own immune system, but generallyrange from about 0.1 mg antibody per kg body weight to about 10 mgantibody per kg body weight, preferably about 0.3 mg antibody per kg ofbody weight to about 5 mg of antibody per kg of body weight. In view ofthe minimization of extraneous substances and the lower probability of“foreign substance” rejections which are achieved by the presentcanine-like and antibodies of this invention, it may be possible toadminister substantial excesses of these antibodies.

The dosage administered will, of course, vary depending upon knownfactors such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms kind of concurrenttreatment, frequency of treatment, and the effect desired.

As a non-limiting example, treatment of NGF-related pathologies in dogscan be provided as a biweekly or monthly dosage of anti-NGF antibodiesof the present invention in the dosage range described above. Exampleantibodies for canine therapeutic use are high affinity (these may alsobe high avidity) antibodies, and fragments, regions and derivativesthereof having potent in vivo anti-NGF activity, according to thepresent invention. Single or multiple administrations of thecompositions can be carried out with dose levels and pattern beingselected by the treating veterinarian. In any event, the pharmaceuticalformulations should provide a quantity of the antibody(ies) of thisinvention sufficient to effectively treat the subject.

Diagnostic Applications

The present invention also provides the above anti-NGF antibodies andpeptides for use in diagnostic methods for detecting NGF in caninesknown to be or suspected of having an NGF related disorder. In anembodiment of the invention the NGF related disorder is pain. In anotherembodiment the NGF related disorder is osteoarthritis Anti-NGFantibodies and/or peptides of the present invention are useful forimmunoassays which detect or quantitate NGF, or anti-NGF antibodies, ina sample. An immunoassay for NGF typically comprises incubating aclinical or biological sample in the presence of a detectably labeledhigh affinity (or high avidity) anti- NGF antibody or polypeptide of thepresent invention capable of selectively binding to NGF, and detectingthe labeled peptide or antibody which is bound in a sample. Variousclinical assay procedures are well known in the art. See, ex.IMMUNOASSAYS FOR THE 80'S (Voller et al., eds., Univ. Park, 1981). Suchsamples include tissue biopsy, blood, serum, and fecal samples, orliquids collected from animal subjects and subjected to ELISA analysisas described below. Thus, an anti-NGF antibody or polypeptide can befixed to nitrocellulose, or another solid support which is capable ofimmobilizing cells, cell particles or soluble proteins. The support canthen be washed with suitable buffers followed by treatment with thedetectably labeled NGF specific peptide, antibody or antigen bindingprotein. The solid phase support can then be washed with the buffer asecond time to remove unbound peptide or antibody. The amount of boundlabel on the solid support can then be detected by known method steps.

“Solid phase support” or “carrier” refers to any support capable ofbinding peptide, antigen, or antibody. Well-known supports or carriers,include glass, polystyrene, polypropylene, polyethylene,polyvinylidenefluoride (PVDF), dextran, nylon, amylases, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble to some extent or insolublefor the purposes of the present invention. The support material can havevirtually any possible structural configuration so long as the coupledmolecule is capable of binding to NGF or an anti-NGF antibody. Thus, thesupport configuration can be spherical, as in a bead, or cylindrical, asin the inside surface of a test tube, or the external surface of a rod.Alternatively, the surface can be flat, such as a sheet, culture dish,test strip, etc. For example, supports may include polystyrene beads.Those skilled in the art will know many other suitable carriers forbinding antibody, peptide or antigen, or can ascertain the same byroutine experimentation. Well known method steps can determine bindingactivity of a given lot of anti-NGF peptide and/or antibody or antigenbinding protein. Those skilled in the art can determine operative andoptimal assay conditions by routine experimentation.

Detectably labeling an NGF-specific peptide and/or antibody can beaccomplished by linking to an enzyme for use in an enzyme immunoassay(EIA), or enzyme-linked immunosorbent assay (ELISA). The linked enzymereacts with the exposed substrate to generate a chemical moiety whichcan be detected, for example, by spectrophotometric, fluorometric or byvisual means. Enzymes which can be used to detectably label theNGF-specific antibodies of the present invention include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta)-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. By radioactively labeling the NGF-specificantibodies, it is possible to detect NGF through the use of aradioimmunoassay (RIA). See Work et al., LAB. TECHNIQUES & BIOCHEM. INMOLEC. BIO(No. Holland Pub. Co., NY, 1978). The radioactive isotope canbe detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography. Isotopes which areparticularly useful for the purpose of the present invention include:³H, ¹²⁵I, ¹³¹I, ³⁵S, and ¹⁴C.

It is also possible to label the NGF-specific antibodies with afluorescent compound. When the fluorescent labeled antibody is exposedto light of the proper wave length, its presence can then be detecteddue to fluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. TheNGF-specific antibodies or antigen binding proteins can also bedelectably labeled using fluorescence-emitting metals such a ¹²⁵Eu, orothers of the lanthanide series. These metals can be attached to the NGFspecific antibody using such metal chelating groups asdiethylenetriaminepentaacetic acid (DTPA) or ethylenediamine-tetraaceticacid (EDTA).

The NGF-specific antibodies also can be detectably labeled by couplingto a chemiluminescent compound. The presence of the chemiluminescentlylabeled antibody is then determined by detecting the presence ofluminescence that arises during the course of a chemical reaction.Examples of useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound can be used to label theNGF-specific antibody, portion, fragment, polypeptide, or derivative ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Detection of the NGF-specific antibody, portion, fragment, polypeptide,or derivative can be accomplished by a scintillation counter, forexample, if the detectable label is a radioactive gamma emitter, or by afluorometer, for example, if the label is a fluorescent material. In thecase of an enzyme label, the detection can be accomplished bycolorometric methods which employ a substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

For the purposes of the present invention, the NGF which is detected bythe above assays can be present in a biological sample. Any samplecontaining NGF may be used. For example, the sample is a biologicalfluid such as, for example, blood, serum, lymph, urine, feces,inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissueextract or homogenate, and the like. The invention is not limited toassays using only these samples, however, it being possible for one ofordinary skill in the art, in light of the present specification, todetermine suitable conditions which allow the use of other samples.

In situ detection can be accomplished by removing a histologicalspecimen from an animal subject, and providing the combination oflabeled antibodies of the present invention to such a specimen. Theantibody (or portion thereof) may be provided by applying or byoverlaying the labeled antibody (or portion) to a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of NGF but also the distribution of NGF in theexamined tissue. Using the present invention, those of ordinary skillwill readily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

The antibody, fragment or derivative of the present invention can beadapted for utilization in an immunometric assay, also known as a“two-site” or “sandwich” assay. In a typical immunometric assay, aquantity of unlabeled antibody (or fragment of antibody) is bound to asolid support that is insoluble in the fluid being tested and a quantityof detectably labeled soluble antibody is added to permit detectionand/or quantification of the ternary complex formed between solid phaseantibody, antigen, and labeled antibody.

The antibodies may be used to quantitatively or qualitatively detect theNGF in a sample or to detect presence of cells that express the NGF.This can be accomplished by immunofluorescence techniques employing afluorescently labeled antibody (see below) coupled with fluorescencemicroscopy, flow cytometric, or fluorometric detection. For diagnosticpurposes, the antibodies may either be labeled or unlabeled. Unlabeledantibodies can be used in combination with other labeled antibodies(second antibodies) that are reactive with the antibody, such asantibodies specific for canine immunoglobulin constant regions.Alternatively, the antibodies can be directly labeled. A wide variety oflabels may be employed, such as radionuclides, fluors, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, ligands (particularlyhaptens), etc. Numerous types of immunoassays, such as those discussedpreviously are available and are well known to those skilled in the art.Importantly, the antibodies of the present invention may be helpful indiagnosing an NGF related disorder in canines. More specifically, theantibody of the present invention may identify the overexpression of NGFin companion animals. Thus, the antibody of the present invention mayprovide an important immunohistochemistry tool. The antibodies of thepresent invention may be used on antibody arrays, highly suitable formeasuring gene expression profiles.

Kits

Also included within the scope of the present invention are kits forpracticing the subject methods. The kits at least include one or more ofthe antibodies of the present invention, a nucleic acid encoding thesame, or a cell containing the same. An antibody of the presentinvention may be provided, usually in a lyophilized form, in acontainer. The antibodies, which may be conjugated to a label or toxin,or unconjugated, are typically included in the kits with buffers, suchas Tris, phosphate, carbonate, etc., stabilizers, biocides, inertproteins, e.g., serum albumin, or the like. Generally, these materialswill be present in less than 5% wt. based on the amount of activeantibody, and usually present in total amount of at least about 0.001%wt. based again on the antibody concentration. Frequently, it will bedesirable to include an inert extender or excipient to dilute the activeingredients, where the excipient may be present in from about, 1% to 99%wt. of the total composition. Where a second antibody capable of bindingto the primary antibody is employed in an assay, this will usually bepresent in a separate vial. The second antibody is typically conjugatedto a label and formulated in an analogous manner with the antibodyformulations described above. The kit will generally also include a setof instructions for use.

The invention will now be described further by the non-limiting examplesbelow.

EXAMPLES

The present invention is further illustrated and supported by thefollowing examples. However, these examples should in no way beconsidered to further limit the scope of the invention. To the contrary,one having ordinary skill in the art would readily understand that thereare other embodiments, modifications, and equivalents of the presentinvention without departing from the spirit of the present inventionand/or the scope of the appended claims.

Example 1 Identification of Mouse Monoclonal Antibody Recognizing CanineNerve Growth Factor (NGF)

A series of murine mAbs were produced against mature, human β-NGF (U.S.Pat. No. 7,727,527). Once these mAbs were generated, they were subjectedto a variety of analyses, including epitope mapping and in vitro testsfor 1) binding to NGF and 2) ability to block binding of NGF to itsreceptors TrkA and to a lesser extent p75 and 3) assessment offunctional antagonism associated with NGF blockade (blockade of TrkAautophosphorylation, blockade of NGF-dependent survival of neurons).Based on these results, it was found that mAbs that bind to variableregions 1 (β-hairpin turn A′→A″), 4 (β-strands C and D) and thecarboxy-terminus are capable of blocking TrkA and p75 binding. Molecularmodeling of the binding epitope on NGF of one of these murine mAbs,RN911, is shown in FIG. 6A. RN911 was shown to be efficacious inpreclinical rodent models of pain associated with arthritis, cancer,surgical incision as well as neuropathic pain and visceral pain.

Anti-NGF Therapy for Joint Pain in Dogs

Amino acid sequence identity is 100% conserved within the bindingepitope of RN911 on B-NGF across dogs, mice and humans (FIG. 6B, above).High affinity binding of RN911 to recombinant canine NGF (K_(D)=755 pM)was experimentally verified and efficacy of the anti-NGF mAb in a caninesynovitis pain model that is routinely used by one skilled in the art inevaluating pain models and described below, was evaluated using RN911.RN911 was produced in sufficient quantities using techniques of oneskilled in the art, for both pharmacokinetics/toleration and efficacystudies in purpose-bred beagles. An efficacy study was designed thatinvolved induction of synovitis at 3 days and 7 days after IV injectionof RN911 and evaluation of lameness/pain at multiple timepointspost-synovitis induction. A pharmacokinetics/toleration study, describedin the next section, was performed prior to the efficacy study to gainconfidence in the safety of RN911 in beagles and to collect data thatwould help guide dose selection for the efficacy study.

DMPK Metabolism and Disposition

As with all proteins, it can be assumed that RN911 is catabolized toamino acids and other endogenous components. Unchanged RN911 is unlikelyto be excreted.

Pharmacokinetics

The pharmacokinetics (PK) of murine anti-NGF mAb RN911 in dogs werestudied following single IV administration . Three dogs were dosed withplacebo, 4 dogs at 1.2 mg/kg, 4 dogs at 3.5 mg/kg, and 1 dog at 9.8mg/kg. Only one dog was utilized at the highest dose. Thepharmacokinetic data exhibited low variability and the similar shape ofthe profiles of ‘free’ RN911 at all doses suggested that target-mediateddisposition was not a factor in the PK. The data exhibited excellentdose proportionality. T₁₁₂ was similar at all doses, averaging 102±27hours (n=9). Clearance, at 0.00051±0.00005 L/h/kg, was very slow. Volumeof distribution, at 0.072±0.021 L/kg, was close to what would beexpected for a protein which is confined to the blood volume of the dog.FIG. 7 shows data from serum analysis of markers analyzed from thesynovitis efficacy study described below. It is clear that the serumconcentrations and pharmacokinetics at 3 mg/kg in the efficacy studywere nearly identical to the values at 3.5 mg/kg in the PK study.

“Bound” RN911 was assayed in both studies using an ELISA method (FIG. 8)The concentrations are reported as NGF-equivalents. The typicalRN911-NGF concentrations, which remained near peak levels for at leastone week, were approximately 0.5 ng/mL in NGF-equivalents. This isroughly 10-fold higher than endogenous NGF concentrations in canineserum (˜40-60 pg/mL).

The typical RN911-NGF serum concentration, when converted toRN911-equivalents, was approximately 5 ng/mL (0.5 ng/mL*molecular weightratio, 150,000/13,425). This is far below the measured free RN911concentrations shown in the first figure and suggest that the mAb mayremain in excess (as compared with NGF concentrations) for several weeksat doses≧3 mg/kg.

Immunogenicity

Anti-drug antibody (ADA) titers were determined before dosing and at theend of the study. As shown in Table 1, there was evidence of a rise inADA titers in at least two of the beagles (see dogs 8 and 12). It is notsurprising that RN911 would generate an immune response in beagles giventhat it is a murine mAb; no attempt was made to perform repeated dosingstudies with this mAb.

TABLE 1 Table 1. Anti-drug antibody (ADA) titers after IV injection ofRN911 ADA Titer (−fold dilution) Dog Dose (mg/kg) Day 0 Day 28 Controlsample — 400 800 1 0 100 <50 2 0 50 50 3 0 <50 <50 5 1.2 50 200 6 1.2 5050 7 1.2 <50 50 8 1.2 50 ≧1600 9 3.5 <50 50 10  3.5 <50 200 11  3.5 <50100 12  3.5 50 800 13  9.8 <50 <50

Bioanalytical Assay Methodology

The free RN911 ELISA was based on the use of recombinant canine NGF asthe capture agent and peroxidase-conjugated donkey anti-mouse IgG as thedetection antibody. QCs as well as standards were included in the assayrun.

The bound RN911-NGF ELISA was based on the use of biotinylated rabbitanti-human NGF adsorbed on a strepavidin plate. This antibody capturedboth NGF-RN911 and free NGF, but the NGF-RN911 was selectively detectedusing peroxidase-conjugated donkey anti-mouse IgG. Standards wereprepared by incubating a known concentration of canine NGF with a slightmolar excess of RN911.

The anti-drug antibody ELISA utilized RN911 as the capture agent. ADAsin the dog serum samples were captured on the plate and detected using amixture of peroxidase-conjugated goat anti-dog antibodies which wereable to detect all four canine IgG subclasses.

Pharmacokinetics/Pharmacodynamics (PK/PD)

PK/PD data were generated with RN911 in the target species in asynovitis efficacy study. In the synovitis pain model, transientinflammation of the synovial membrane in a single stifle is induced viaintra-articular injection of bacterial lipopolysaccharide (LPS).Quantifiable lameness occurs within 2h of synovitis induction, peaks at3-4h, is waning by 6h and is fully resolved after 24h. From an IACUCperspective, it is acceptable to induce synovitis on two separateoccasions (i.e., once in each stifle), provided the lameness associatedwith the first synovitis induction has fully resolved. Therefore wedesigned a study in which synovitis was induced in the same beagle 3days and 7 days after IV treatment (RN911 or PBS vehicle). After eachsynovitis induction, lameness was quantified using a visual analoguescale (VAS).

The results of this study show that RN911 (3 mg/kg, IV) caused astatistically significant decrease in synovitis-associated lameness at 3days after dosing (FIG. 4). At this time, the free RN911 serumconcentrations averaged 24400 ±3900 ng/mL (170±27 nM; FIG. 2). RN911 waseven more effective at 7 days (FIG. 9) when the free RN911 serumconcentration averaged 13200 ±3500 ng/mL (91±24 nM; FIG. 7).

Example 2 Caninization Strategy

The generation of anti-drug antibodies (ADAs) can be associated withloss of efficacy for any biotherapeutic protein including monoclonalantibodies. Comprehensive evaluation of the literature has shown thatspeciation of monoclonal antibodies can reduce the propensity for mAbsto be immunogenic although examples of immunogenic fully human mAbs andnon-immunogenic chimeric mAbs can be found. To help mitigate risksassociated with ADA formation for the mouse anti-NGF, RN911, monoclonalantibody provided herein, a caninization strategy was employed. Thiscaninization strategy is based on identifying the most appropriatecanine germline antibody sequence for CDR grafting (FIG. 3). Followingextensive analysis of all available canine germline sequences for boththe heavy and light chains, germline candidates were selected on theirhomology to RN911, and the CDRs from RN911 were used to replace nativecanine CDRs. The objective was to retain high affinity and cell-basedactivity using fully canine frameworks to minimize the potential ofimmunogenicity in vivo. Caninized mAbs were optimized for mammalianexpression, expressed and characterized for their ability to bind NGFvia SPR. These results are described below in Example 3. Only mAbs thatretained both reliable expression levels and the ability to bind NGFfollowing caninization were advanced for further characterization. ThosemAbs that did not express transiently or lost the ability to bind NGFwere systematically dissected to identify: 1) the chain responsible forloss of function of lack of expression, 2) the framework responsible forthe loss of expression or function and 3) the amino acid(s) responsiblefor loss of expression or function.

Example 3 Caninization of RN911 Antibody

Synthetic constructs representing the caninized variable heavy and lightchains of mAb RN911 were made. Following subcloning of each variablechain into plasmids containing the respective canine heavy or kappaconstant region, plasmids were co-transfected for antibody expression inHEK 293 cells. The canine heavy chain constant region of the presentinvention are not limited to any particular subtype, however in someembodiments the canine heavy chain is described as SEQ ID. NO. 45 or SEQID NO. 48. The canine kappa light chain constant regions are not limitedto any particular sequences, however in some embodiments of the presentinvention the canine kappa constant region is described as SEQ ID. NO.47. Frameworks used for mAb “canE3M65” expressed in HEK 293 transientexpression system and retained NGF binding upon caninization. (Seq IDNO.33 “CAN-E3M-VL”, SEQ ID NO. 34. “CAN-N2G9-VH”).

In contrast, the germline sequences used for the can618 (CAN-618-VL;CAN-N2G9-VH) caninization efforts resulted in certain non-expressingmAbs. Chimeric, heterochimeric, and caninized versions of mAb RN911 wereexpressed and characterized for their ability to bind canine NGF viaSPR. These results demonstrated that the caninized antibody did notexpress. Also, with respect to the heterochimeras, the chimeric heavychain paired with the caninized light chain lost expression, while thecaninized heavy chain paired with the chimeric light chain retainedexpression in HEK293 cells. Based on the results obtained from theheterochimeras, it was deduced that the caninized light chain wasresponsible for the loss of expression. In an effort to restoreexpression of the caninized versions of RN911 in which expression waslost, the caninized light chain was modified by swapping frameworksequences. FIG. 10 provides an overview of the can911 light chainframework substitution work. This work identified an antibody replacingresidues within the canine framework II (FWII) with mouse framework IIsequence and restoring expression in HEK293 cells (SEQ ID NO.35“CAN-618-VL”, SEQ ID NO. 36 “CAN-QC23-VL”, and variable heavy chain SEQID NO.34 “CAN-N2G9-VH”).

FIG. 11 summarizes the results of both the expression and respectivemutations. These data demonstrate that the caninized derivatives usingframeworks 3 and 4 both retain HEK expression levels comparable to theirprecursor molecule and show binding to canine NGF. Framework 1 retainedexpression to a lesser extent, but also maintained binding to canineNGF.

Example 4 Characterization of Canine NGF Binding to Caninized Anti-NGFmAbs

Affinities of each caninized anti-NGF antibody to canine NGF weremeasured using SPR (Surface Plasmon Resonance). In addition, afunctional in vitro assay was developed to measure inhibition constantsfor the mAbs ability to inhibit binding of NGF to TrkA. Data shown inFIGS. 12 and 13 for can6512 (CAN-N2G9-VH, CAN-E3M-VL, CAN-65E-HC,CAN-KAPPA-LC) and RN911 illustrate high affinity binding of mAbs to NGFand potent inhibition by mAbs of NGF binding to receptor trkA. Someaffinity was lost, however, upon caninization to mAb CAN-N2G9-VH,CAN-E3M. Due to this loss of affinity, the caninized mAb was affinitymatured (see Example 5 for details) to regain potency. Included in FIGS.12 and 13 are the results of the affinity matured products, SSM57 (SEQID NO. 27 “CAN-SSM57-VH”, SEQ ID NO. 26 “CAN-SSME3M-VL”), SSM58 (SEQ IDNO. 28 CAN-SSM58-VH, SEQ ID NO. 26 “CAN-SSME3M-VL”), and SSM66 (SEQ IDNO.29 CAN-SSM66, SEQ ID NO. 26 CAN-SSME3M-VL) on each SPR assay.

Example 5 Affinity Maturation of Caninized Anti-NGF mAb

Affinity maturation of canE3M65 (SEQ ID NO. 16 “CAN-E3M-VL”, SEQ ID NO.17 “CAN-N2G9-VH”) was necessary to return the affinity of the caninizedmAb to that of the progenitor mouse antibody. Two antibody librarieswere designed to contain individual point mutations within the antibodysequence in CDR regions only. FIG. 15 outlines the design strategy ofeach Fab library in which the Look-Through Mutagenesis (LTM) librarycomprises individual site mutations limited to one of ninerepresentative amino acid residues, while the Site-SaturationMutagenesis (SSM) library can sample any natural amino acid (Cys and Metwere excluded here). The LTM library was constructed to identifybeneficial mutations in CDRH3, CDRL1, and CDRL3. Alternatively, the SSMlibrary mutations are located in CDRH1, CDRH2, and CDRL2, allowing us tocombine beneficial mutations from each library.

The LTM library was constructed from a series of primers designed usingan oligonucleotide design program commonly used by those of skill in theart. PCR products were combined to achieve a Fab library comprised ofsingle mutations on each of the three specified CDRs, as well ascombinations including variations on CDRH3, CDRL1, and/or CDRL3. A 93%ligation efficiency was achieved with a library size of ˜10̂4. Fourrounds of panning with decreasing amounts of biotinylated canine NGFwere run and extended off-rates were selected for by extended overnightwash steps. Amount of NGF antigen used ranged from 1 ng/μL beads to 10pg/μL beads and Fabs were eluted using non-biotinylated NGF in excess.After the fourth round of panning, outputs were cloned into a TOPOvector and sequenced. Enriched mutations are shown in Table 2.

TABLE 2 Frequency of mutation; HC CDR3 98 99 100 101 102 103 104 105 106107 108 109 110 E3 G G Y W Y A T S Y Y F D Y 911 G G Y Y Y G T S Y Y F DY A(1) K(4) D(5) S(4) W(3) S(3) W(2) A(1) S(3) K(3) D(2) S(2) D(2) H(2)V(1) W(2) S(2) K(2) Y(2) Q(1) A(2) P(2) Q(2) W(1) L(2) H(1) P(1) D(2)W(1) Q(1) Q(1) A(1) D(1) L(1) P(1) P(1) P(1) Q(1) H(1) L(1) H(1) A(1)H(1) LC CDR1 24 25 26 27 28 29 30 31 32 33 34 E3 R A S Q S I S N N L N911 R A S Q D I S N H L N P(1) G(2) N(1) P(2) S(2) H(1) P(3) H(3) K(2)Q(2) A(2) Q(1) A(1) G(1) Y(1) Y(1) D(3) Y(1) D(1) 5(1) R(1) N(1) S(1)T(1) S(2) S(1) H(1) W(1) A(1) Q(1) P(2) P(1) Y(1) P(1) K(2) N(1) P(1)D(1) Q(1) N(1) L(1) K(1) A(1) LC CDR3 89 90 91 92 93 94 95 96 97 E3 Q QE H T L P Y T 911 Q Q S K T L P Y T K(2) D(3) P(2) K(1) S(1) A(1) Y(2)Q(1) K(1) E(1) K(1) D(1) H(1) A(1) Y(1) A(1) I(1) Y(1) L(1) W(1) Y(1)P(1) P(1) H(1) Q(1) L(1) Y(1) H(1) E(1) W(1)

Indicated constructs were converted into full IgGs and expressedtransiently in HEK293 cells. Results of expression for individual andcombined mutations are shown below in Tables 3 and 4

SPR on the supernatants were performed as an initial screen, goodbinders were purified, and pure mAb was again run via SPR to measurebinding to canine NGF (TABLE 5). Constructs LTM109 and LTM135 werechosen to progress based on binding affinities.

TABLE 5 BiacoreT100: Dog NGF and Abs Binding Kinetics Summary 11/8 11/2211/24 11/29 Name HC LC K_(D) (M) K_(D) (M) K_(D) (M) K_(D) (M) CommentshE3 Y101W,G103A D28S, H32N, SK91EH 1.29E−12 1.12E−12 3.37E−12 1.49E−12CanE3M 65112 3.35E−09 1.46E−09 2.35E−09 3.51E−09 32 Y100W D28S 2.25E−0933 Y100W *D28S,NH31HN no binding no binding 34 Y100W D28S, H32N 6.39E−108.85E−10 ✓/? 35 Y100W D28S, H32N, K92H 2.37E−10 8.10E−10 ✓ 36 Y100W K92D5.50E−09 37 Y100W *D28S, NH31HN, S91Y 1.91E−09 38 Y100W D28S, NH31HN,K92D 2.15E−09 39 Y100W D28S, S91Y no binding no binding 40 Y100W D28S,K92D weak binding weak binding 41 Y100W D28S, H32N, SK91EH weak bindingweak binding 43 Y100W K92H 5.67E−09 44 Y100W S91E no binding 45 Y100WH32N 2.10E−09 4.35E−09 109 Y101W, G103A D28S, H32N, K92H 5.15E−107.48E−10 ✓ 110 Y101W, G103A K92D 3.26E−12 9.42E−10 Agg? 111 Y101W, G103A*D28S, NH31HN, S91Y 1.46E−10 2.88E−11 Agg? 112 Y101W, G103A D28S,NH31HN, K92D 1.65E−12 1.52E−11 Agg? 133 G103A K92H 1.33E−12 1.10E−11Agg? 135 G103A H32N 1.38E−10 6.10E−12 Agg?

Various KD's correspond to different sensors, surface density variesslightly. Agg indicates that a binding curve showing signs of antibodyaggregation was seen in at least one run.

Four rounds of phage display using the SSM library were performed usingsingle site mutations on the same wild-type sequence used for LTM.Libraries were panned against biotinylated or free canine NGF andselected for high affinity and slow off-rate after each round.Selections were run as shown below, round 4 outputs were TOPO cloned andsequenced.

Site-Saturation Mutagenesis for NGF Affinity Maturation—SelectionStrategy

Samples used:

-   (1) negative control using streptavidin beads w/o antigen-   (2) bioNGF antigen on streptavidin beads.-   (3) Immunotube with dNGF immobilized on tube-   (4) E33 positive control on streptavidin beads with bioNGF-   (5) E33 positive control with immunotube/dNGF antigen

*note positive controls will verify appropriate stringency

Note: Using chemical elution only (100 mM TEA) to assure that we don'tscreen out the tightest binders.Negative selection for immunotube=tube w/o dNGF immobilized.Negative selection for strep beads=beads w/o bioNGF immobilized.extended washes select for very slow off-rates; stringency increasedwith rounds.

Round 1: Samples (1), (2), (3)

Immunotube: 50 ug/mL dNGF

Block 2 hours Negative selection 1 hour Bind 1 hour Wash 10x PBST wash #4 1 hour 10x PBS wash #17 2 hours Elute 30 minStrep Beads: 100 nM bioNGF (negative control=PBS only)

Block phage 1 hour Block beads 2 hours Negative selection 1 hour Bindantigen/phage 1 hour Bind complex/beads 15 min Wash 6x MPBST wash #4 1hour 6x MPBST wash #11 2 hours 2x PBS * each line represents transfer tonew tube Elute 5 min

Round 2: Samples (1), (2), (3), (4), (5)

Immunotube: 20 ug/mL dNGF

Block 2 hours Negative selection 1 hour Bind 1 hour Wash 15x PBST wash #4 2 hours 15x PBS wash #25 2.5 hours Elute 30 minStrep Beads: 50 nM bioNGF (negative control=PBS only)

Block phage 1 hour Block beads 2 hours Negative selection 1 hour Bindantigen/phage 1 hour Bind complex/beads 15 min Wash 10x MPBST wash #4 2hours 10x MPBST wash #18 2 hours 4x PBS PBS wash #2 30 min * each linerepresents transfer to new tube Elute 5 min

Round 3: Samples (1), (2), (3), (4), (5)

Immunotube: 20 ug/mL dNGF

Block 2 hours Negative selection 1 hour Bind o/n Wash 15x PBST wash # 42 hours 15x PBS wash #25 6 hours Elute 30 minStrep Beads: 30 nM bioNGF (negative control=PBS only)

Block phage 1 hour Block beads 2 hours Negative selection 1 hour Bindantigen/phage 1 hour Bind complex/beads o/n Wash 10x MPBST wash #4 2hours 10x MPBST wash #18 30 min 4x PBS PBS wash #2 6 hours * each linerepresents transfer to new tube Elute 5 min

Round 4: Samples (1), (2), (3), (4), (5)

Immunotube: 10 ug/mL dNGF

Block 2 hours Negative selection 1 hour Bind 1 hour Wash 20x PBST wash #4 3 hours 20x PBS wash #32 13 hours Elute 30 minStrep Beads: 15 nM bioNGF (negative control=PBS only)

Block phage 1 hour Block beads 2 hours Negative selection 1 hour Bindantigen/phage 1 hour Bind complex/beads 15 min Wash 15x MPBST wash #4 3hours 15x MPBST 5x PBS PBS wash #2 13 hours * each line representstransfer to new tube Elute 5 min

Table 6 above shows the sequenced outputs from SSM-library display aswell as initial diversity. Enriched sites were selected for bothsingle-site and multi-site (combinations of enriched sites) mutationsand subsequent IgG conversion. SSM mutations for both heavy and lightchains were generated on three templates: (1) wild-type mAb, (2) LTM109(3) LTM135.

Mutated antibodies were expressed in HEK293 cells and evaluated forbinding affinities to canine NGF (results below; expression results inTables 7-9). Antibodies SSM57 (SEQ ID NO. 27 “CAN-SSM57-VL, SEQ ID NO 26“CAN-SSM-E3M-VL”), SSM58 (SEQ ID NO. 28 “CAN-SSM58-VH”, SEQ ID NO. 26“”CAN-SSM-E3M-VL”), and SSM66 (SEQ ID NO. 29 “CAN-SSM66-VH”, SEQ ID NO.26 “CAN-SSM-E3M-VL”) showed highest affinities.

TABLE 9 ka Ligand Samples Fit (M−1 s−1) kd (s−1) KD (M) A1 HBS-EP 1:1Binding 1.39E+07 6.61E−08 no binding A2 CanE3M 1:1 Binding 1.72E+048.11E−05 4.70E−09 65112 A3 hE3 1:1 Binding 8.75E+04 1.67E−07 1.91E−12 A4LTM135 1:1 Binding 2.51E+04 4.23E−05 1.69E−09 A5 SSM-2 1:1 Binding8.08E+03 1.14E−04 1.42E−08 A6 SSM-3 1:1 Binding 4.04E+03 1.67E−044.14E−08 A7 SSM-8 1:1 Binding 4.13E+03 1.73E−04 4.18E−08 A8 SSM-12 1:1Binding 9.57E+02 4.13E−03 4.32E−06 A9 SSM-13 1:1 Binding 7.75E+032.41E−03 3.11E−07 A10 SSM-14 1:1 Binding 3.44E+05 8.12E−03 2.36E−08 A11SSM-17 1:1 Binding 2.03E+04 1.40E−03 6.91E−08 A12 SSM-19 1:1 Binding4.38E+04 1.64E−03 3.74E−08 A13 SSM-22 1:1 Binding 1.19E+03 2.96E−042.49E−07 A14 SSM-34 1:1 Binding 6.24E+04 1.92E−04 3.08E−09 A15 SSM-361:1 Binding 8.20E+03 3.30E−04 4.02E−08

Example 6 Production of Caninized Antibodies Form Glutamine Synthetase(GS) Plasmids

The genes encoding the caninized RN911 mAbs were cloned into GS plasmidspEE 6.4 and pEE 12.4, respectively (Lonza, Basel, Switzerland). Theresulting plasmids were digested according to the manufacturer'sprotocol and ligated together to form a single mammalian expressionplasmid. Each plasmid was used to transfect HEK 293 cells and expressionwas carried out in 20 L of culture media. Protein was isolated formconditioned HEK medium using Protein A affinity chromatography accordingto standard protein purification methods. Medium was loaded ontochromatographic resin and eluted by pH shift. Eluted protein was pHadjusted, dialyzed, and sterile filtered prior to use. The resultingantibody was greater than 99 percent monomeric by analytical sizeexclusion chromatography with no high molecular weight aggregatesobserved. This antibody was subsequently used for evaluation in the dogsynovitis model to evaluate in vivo efficacy.

Example 7 Evaluation Of Caninized Antibody in the Dog Synovitis Model

Caninized mAb (CAN-SSME3M-VL, CAN-SSM57-HC) was tested in dog synovitismodel at 5 mg/kg IV and 5 mg/kg SC. Results of study are shown in FIG.14, inducing synovitis at 7 days and 28 days post-dose. Caninized mAbshowed efficacy for both SC and IV dosing regimens both 7 and 28 dayspost-dose.

Example 8 Pharmacokinetics of Caninized Antibody

The pharmacokinetics (PK) of the caninized SSM57 mAb (PF-06442591) wasstudied in six dogs at 5 mg/kg using subcutaneous (SC) administration.Dogs were dosed with mAb at 5 mg/kg SC once every 14 days (Day 0, Day14, Day 28) for a total of three injections and were euthanized on Day35 for a full safety assessment (see Safety information below). Serumsamples from each dog were collected prior to each dose and periodicallyover 35 days. Antibody concentration was measured in an ELISA basedformat in which canine NGF was used to capture mAb from the serum. Theantibody was then detected using a labeled anti-dog antibody recognizingthe Fc portion of the antibody subclass. Results are shown in FIG. 16.

Example 9 Evaluation of Caninized Antibody in Rat MIA Model

Osteoarthritis (OA) is a degenerative joint disease characterized byjoint pain and a progressive loss of articular cartilage.Intra-articular injection of MIA induces loss of articular cartilagewith progression of subchondral bone lesions that mimic those of OA.This model offers a rapid and minimally invasive method to reproduceOA-like lesions in a rodent species.

The analgesic effect of caninized anti-NGF antibodies at one dose of MIAin the rat MIA model of osteoarthritis was demonstrated by dosingcaninized SSM57 mAb (PF-06442591) twice during the study on study day 7and study day 14. Pain was assessed using weight bearing test forsustained pain and joint compression (Randall Selitto) test formechanical hyperalgesia. See FIG. 17 for a schematic of the rat MIAprocedure.

TABLE 10 CONSTITUTION OF TEST GROUPS AND DOSE LEVELS The table lists theexperimental groups compared in the study and respective dose levels.Group Group Dose Dosing Testing No. Size Treatment Volume (mg/kg) RouteRegimen Regimen 1 n = 10 Vehicle 1.6 ml/kg 0 SC Once on study Weightbearing test days on study days 7 and 14 −1, 9, 16, 21 and 28. 2 N = 10Positive   5 ml/kg 10 SC Once on study Randall-Selitto test Control dayson study days −1, 20 (Morphine) 9 and 16 1 hour and 28. pre weightbearing testing 3 N = 10 mAb1 1.6 ml/kg 8 SC Once on study 4 N = 10 mAb21.6 ml/kg 8 SC days 7 and 14

Sensitizing Materials Preparation:

A concentration of 60 mg/ml of MIA solution was prepared in saline. Eachrat was dosed with 50 μl of prepared solution i.e. 3 mg of MIA.

Vehicle Preparation (Group 1):

The Vehicle control was saline.

Morphine Preparation at a dose of 10 mg/kg (Group 2):

-   -   1. Added 13.5 ml saline to 1.5 ml morphine.    -   2. Injected 1 ml of received solution per rat weighing 200 g.

Test Items Preparation (Groups 3-4):

Antibodies were 5 mg/ml. The compounds were stored at 2-8° C. andprotected from light prior to use. No vigorous shaking. The compoundswere warmed to room temperature for 1 hour prior to dosing.

Treatment:

Vehicle (Group 1) and Test Item mAb s (Groups 3 and 4) were administeredonce via SC on testing days 7 and 14.

Morphine (Group 2), the positive control in this study, was administeredonce via SC on the testing days 7 and 14.

Routes of Administration

i. Vehicle Control SC ii Positive Control (Morphine) at a dose of 10mg/kg SC iii. Test Item mAb SC

Statistics/Evaluation

All data are presented as means ±SEM. Each treatment group was comparedto vehicle group using a one way ANOVA test followed by Tukey test(Prism V 4.0, GraphPad Software). Comparisons between vehicle group andmorphine group and within the vehicle group for evaluating the modelwere performed using T-test. A p value<0.05 is considered to represent asignificant difference.

Animal Care and Use

Species/Strain: Rat Ola Wistar.

Gender: Male.

Age/Weight: Young adults; at study initiation (130-180 g).

Animal Health: The health status of the animals used in this study wasexamined upon their arrival. Only animals in good health wereacclimatized to laboratory conditions and used in the study.

Acclimation: At least 5 days.

Housing: During acclimation and throughout the entire study duration,animals were housed within a limited access rodent facility and kept ingroups with a maximum of 3 rats per polypropylene cage. The cages werefitted with solid bottoms and filled with sterile wood shavings asbedding material.

Food and Water: Animals are provided ad libitum with a commercial,sterile rodent diet and had free access to drinking water that issupplied to each cage via polyethylene bottles with stainless steelsipper tubes.

Environment: Automatically controlled environmental conditions were setto maintain temperature at 17-23° C. with a relative humidity (RH) of30-70%, a 12:12 hour light: dark cycle and 15-30 air changes/h in thestudy room. Temperature and RH were monitored daily.

Randomization: During the acclimation period, animals were randomlyassigned to experimental groups. Each dosing group was kept in separatecages to avoid cross-contamination which can occur through consumptionof fecal matter.

Termination: At the end of the study, surviving animals were euthanizedby pentobarbital sodium.

Study was performed following approval of an application form submittedto the Committee for Ethical Conduct in the Care and Use of LaboratoryAnimals that stated that the study complied with the rules andregulations set forth.

Weight Bearing Test: Rat Training—Habituation to Testing Environment:

This training regime was required as it was anticipated that the levelof variability within the experiment is relatively high. Therefore, useof this protocol would help to reduce variability and increase thelikelihood of a successful study. Each rat was handled for 1 min thenplaced in the test apparatus (incapacitance meter) for 5 minutes(habituation to test apparatus). This procedure was performed on studyday −2.

Weight Bearing Assessment Details:

Weight bearing deficits were measured in all rats on study day -1, thismeasurement served as a baseline. Weight bearing was then recorded onstudy days 9, 16, 21 and 28. Incapacitance meter records 3 measurementsover a period of 5 seconds and then averages these to obtain 1 value.

Mechanical Hyperalgesia (Randall-Selitto test):

Mechanical thresholds, expressed in grams, was measured in rats with theRandall-Selitto test using an analgesimeter (Ugo Basile). The test wasperformed by applying a noxious pressure to the hind paw. By pressing apedal that activated a motor, the force increased at a constant rate onthe linear scale. When the animal displayed pain by withdrawal of thepaw or vocalization, the pedal was immediately released and thenociceptive threshold read on a scale. The cut-off of 400 g was used toavoid potential tissue injury. Randall-Selitto test was performed onstudy days -1 (baseline) and 28.

Clinical Signs:

Throughout the 30-day study, careful clinical examinations wereperformed at least once daily. If any abnormalities were observed theywere recorded. Observations include changes in skin, fur, eyes, mucousmembranes, occurrence of secretions and excretions (e.g., diarrhea),autonomic activity (e.g., lacrimation, salivation, piloerection, pupilsize, unusual respiratory pattern), gait, posture, and response tohandling, as well as the presence of unusual behavior, tremors,convulsions, sleep, and coma.

RESULTS:

Mean group body weight for all groups (g).

TABLE 11 Baseline Day 7 Day 14 Day 21 Day 27 Treatment Mean SEM Mean SEMMean SEM Mean SEM Mean SEM 1 Vehicle 178.9 3.04 258.5 4.43 295.3 5.92318.2 7.21 341.4 7.57 2 Morphine 178.5 2.97 256.2 6.12 285.1 7.45 300.69.04 316 9.87 10 mg/kg 5 mAb1 176.8 4.57 258.7 5.98 296.4 7.01 319.2 7.8342.9 7.66 6 mAb2 178 4.35 253.4 5.8 290.7 6.66 311.6 7.07 332.6 7.71Response to Weight bearing test (difference between legs) (g):

*p<0.05 vs. Vehicle using one-way ANOVA followed by Tukey test.

The weight bearing of each leg was measured separately by weight bearingapparatus (IITC, Series 8, Model 600®). This test quantifies thespontaneous postural changes reflecting spontaneous pain byindependently measuring, on two separate sensors, the weight that theanimal applies on each hind paw.

Results were calculated and represented as the percentage of weight thatthe animal leaned on the injected right leg or intact left leg from thetotal amount of leaned weight on the two hind legs. Then the differencebetween the two values of intact left leg minus injected right leg iscalculated.

Weight bearing test measures the animal ability to carry its weight onthe hind legs. In normal condition the animal carries its weight equallyon both hind legs (50% on the right leg and 50% on the left leg).Therefore, the difference between the percentages of weight carried oneach leg will be close to 0%. As the animal experiences pain, thissituation changes. The animal will tend to carry more weight on thenonpainful leg and less weight on the painful leg. As a result, thedifference between the percentages of weight carried on both legsincreases.

1-13: (canceled)
 14. A method of treating a canine for an NGF relateddisorder comprising administering a therapeutically effective amount ofa veterinary composition comprising an antigen binding protein thatspecifically binds to canine Nerve Growth Factor comprising a variableheavy chain comprising SEQ ID NO. 27 and a variable light chaincomprising SEQ ID NO. 26 further comprising a pharmaceuticallyacceptable carrier.
 15. The method of claim 14, wherein the NGF relateddisorder condition is selected from the group consisting of:cardiovascular diseases, atherosclerosis, obesity, type 2 diabetes,metabolic syndrome, pain and inflammation.
 16. The method of claim 15wherein the NGF related disorder is pain.
 17. The method of claim 16wherein the type of pain is selected from the group consisting of:chronic pain, inflammatory pain, post-operative incision pain,neuropathic pain, fracture pain, osteoporotic fracture pain,post-herpetic neuralgia, cancer pain, pain resulting from burns, painassociated with wounds, pain associated with trauma, neuropathic pain,pain associated with musculoskeletal disorders such as rheumatoidarthritis, osteoarthritis, ankylosing spondylitis, seronegative(non-rheumatoid) arthropathies, non-articular rheumatism andperiarticular disorders and peripheral neuropathy.
 18. The method ofclaim 17 wherein the pain is osteoarthritis pain. 19-29: (canceled) 30.The method of claim 14 wherein the antigen binding protein is selectedfrom the group consisting of: a monoclonal antibody; a chimericantibody, a single chain antibody, a tetrameric antibody, a tetravalentantibody, a multispecific antibody, a domain-specific antibody, adomain-deleted antibody, a fusion protein, an ScFc fusion protein, anFab fragment, an Fab′ fragment, an F(ab′)₂ fragment, an Fv fragment, anScFv fragment, an Fd fragment, a single domain antibody, a dAb fragment,a small modular immunopharmaceutical (SMIP) a nanobody, and IgNARmolecule.
 31. The method of claim 30 wherein said antigen bindingprotein is a monoclonal antibody.